Engineered ketoreductases and methods for producing stereoisomerically pure statins

ABSTRACT

The present disclosure provides ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, method of using the engineered ketoreductase enzymes to synthesize a variety of chirally pure compounds, and the chirally pure compounds prepared therewith.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 11/856,696,filed Oct. 1, 2007, now U.S. Pat. No. 7,879,585 B2, and U.S. Ser. No.60/848,951, filed on Oct. 2, 2006, each of which is hereby incorporatedby reference in its entirety.

2. TECHNICAL FIELD

The present disclosure relates to compositions and methods useful forthe stereoselective reduction of hydroxy oxo esters, such as5-hydroxy-3-oxo-hexanoate esters, to yield stereoisomerically purecorresponding syn dihydroxy esters. More specifically, the disclosureconcerns non-naturally occurring engineered ketoreductase enzymes havingimproved properties as compared to the wild-type ketoreductases fromwhich they were derived, polynucleotides encoding the engineeredketoreductase enzymes, host cells comprising such polynucleotides,methods of using the engineered ketoreductase enzymes to synthesize avariety of stereoisomerically pure compounds, and the stereoisomericallypure compounds synthesized therewith.

3. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing submitted concurrently herewith via EFS-WEB under37 C.F.R. §§1.821(c) and 1.821(e) as a CRF having filename376247-012.txt, created on May 30, 2008 and having a file size of 716Kbytes, is herein incorporated by reference in its entirety.

4. BACKGROUND

Stereoisomerically pure syn 3,5-dihydroxyhexanoate esters are key chiralintermediates for the synthesis of cholesterol lowering statins such asatorvastatin calcium (sold under the brand name LIPITOR® by Pfizer,Inc.), rosuvastatin calcium (sold under the brand name CRESTOR® byAstraZeneca, Ltd.), and pitavastatin (sold in Japan under the brand nameLipalo by Kowa Company Ltd. and Nissan Chemical Industries). Whilevarious methods for producing these chiral intermediates are known,including both chemical and enzymatic methods of reducing thecorresponding 5-hydroxy-3-oxohexanoate ester, these methods suffersignificant drawbacks, making them less than ideal for commercial scalesynthesis. Given the importance of these key chiral intermediates in thesynthesis of cholesterol lowering statins, compositions and methodsuseful for synthesizing these compounds in a cost effective andefficient manner would be highly desirable.

5. SUMMARY

As mentioned above, stereoisomerically pure syn 3,5-dihydroxyhexanoateesters according to structural formula (IIa):

wherein X is selected from halo (e.g., chloro and bromo), cyano and—OR⁶, where R⁶ is hydrogen or a protecting group, and R¹ is selectedfrom (C2-C12) alkyl, (C6-C10) aryl and (C7-C12) arylalkyl, are keyintermediates in the synthesis of cholesterol lowering statins,including but not limited to, atorvastatin; rosuvastatin, andpitavastatin. These key chiral intermediates are typically synthesizedby reducing the corresponding enantiomerically pure5-hydroxy-3-oxohexanoate ester according to structural formula (Ia):

under conditions in which the syn dihydroxy ester diastereomer ofstructural formula (IIa), supra, is obtained in diastereomeric excessover the corresponding anti dihydroxy ester diastereomer of structuralformula (IIb):

Recently, certain ketoreductase enzymes have been identified that can beused to carry out this enantioselective reduction (see, e.g., U.S. Pat.No. 6,645,746 and WO 01/40450). The present inventors have discoveredthat ketoreductase enzymes capable of catalyzing the above-describedenantioselective reduction can be engineered using laboratory evolutiontechnologies, such as DNA shuffling, to yield non-naturally occurringengineered ketoreductases that have improved properties as compared tothe naturally-occurring wild-type ketoreductase enzyme obtained fromSaccharomyces cerevisiae. Further, the disclosure also provides isolatednaturally-occurring ketoreductases that comprise any one of the sets ofmutations disclosed herein for the engineered ketoreductases. Thus, suchnaturally-occurring ketoreductases that comprise such sets of mutationscan be used in any one of the processes, kits, reaction mixtures, etc.disclosed herein.

The improved properties can be related to different aspects of thewild-type ketoreductase enzyme. For example, the improved properties caninvolve the stability of the enzyme under the conditions of thestereoselective reduction reaction, the enzymatic activity of theketoreductase enzyme, and/or sensitivity to inhibition (e.g., productinhibition).

Thus, in one aspect, the present disclosure provides engineered (orisolated naturally-occurring variant) ketoreductase enzymes having animproved property as compared to the naturally-occurring wild-typeketoreductase enzyme obtained from Saccharomyces cerevisiae. Theengineered ketoreductases can possess a single improved property, orthey can possess two or more improved properties, in any combination(s).In some aspects, the engineered ketoreductases can comprise an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the wild-type ketoreductase enzyme amino acidsequence, with the proviso that the engineered amino acid sequencecomprises one or more mutations that are sufficient to provide animproved property.

In some embodiments, the engineered ketoreductase enzyme has increasedenzymatic activity as compared to the wild-type ketoreductase enzyme.Improvements in enzymatic activity can be measured by comparing thespecific activity of the engineered ketoreductase with that of thewild-type ketoreductase enzyme using standard enzyme assays. The amountof the improvement can range from 1.5 times the enzymatic activity ofthe corresponding wild-type ketoreductase enzyme, to as much as 2 times.5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times,150 times, 200 times, 250 times, 300 times, or more enzymatic activity.In specific embodiments, the engineered ketoreductase enzyme exhibitsimproved enzymatic activity in the range of 1.5 to 50 times, 1.5 to 100times, 1.5 to 150 times, 1.5 to 200 times, 1.5 to 250 times, or 1.5 to300 times greater than that of the wild-type ketoreductase enzyme.Improvements in enzyme activity also include increases instereoselectivity or reduced product inhibition.

In some embodiments, the engineered ketoreductase enzyme exhibitsimproved thermal stability as compared to the wild-type ketoreductaseenzyme. Improvements in thermal stability can be measured by comparingthe degree of enzymatic activity retained by the engineeredketoreductase enzyme following a specified period of incubation at aspecified temperature with that retained by the corresponding wild-typeenzyme under similar conditions.

The engineered ketoreductase enzymes described herein can be obtained bymutagenizing the gene encoding naturally-occurring wild-typeketoreductase enzyme of Saccharomyces utilizing standard laboratorytechniques, including the various mutagenesis and recombinationtechniques.

In some embodiments, the engineered ketoreductase enzymes describedherein have a sequence that corresponds to the amino acid sequence ofthe wild-type ketoreductase from which they are derived and include oneor more amino acid mutations that result in an improvement in theproperty of the enzyme. In some embodiments, the mutations are aselected combination of mutations that overall improve one or moreproperties of the enzyme. In some embodiments, such as where theimproved property is from a single mutation or specific combination ofmutations, the engineered ketoreductase may optionally include one ormore conservative mutations at other residue positions within thepolypeptide sequence.

In some embodiments, the ketoreductase enzyme comprises an amino acidsequence that corresponds to SEQ ID NO:2, or is at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, andwherein the amino acid sequence comprises at least one mutation over SEQID NO:2 at one of the following residue positions: 6, 27, 31, 36, 48,63, 65, 86, 125, 152, 160, 165, 194, 214, 218, 234, 248, 250, 263, 290,296, 297, 301, and 307. In one embodiment, the at least one mutation atone of the above positions must be a non-conservative mutation ascompared to the wild-type SEQ ID NO:2 sequence.

In some embodiments, the ketoreductase enzyme comprises an amino acidsequence that corresponds to SEQ ID NO:2, or is at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, andwherein the amino acid sequence comprises an aromatic residue atposition 63. In one embodiment, the aromatic residue is tryptophan.

In some embodiments, the ketoreductase enzyme has an amino acid sequencethat is at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identical to any one of the amino acid sequences listed in theSequence Listing, with the proviso that the amino acid sequence is notidentical to the wild-type sequence (SEQ ID NO:2). In some embodiments,the ketoreductase has an amino acid sequence that is identical to anyone of the amino acid sequences listed in the Sequence Listing andincludes one or more conservative mutations (including for example, 1,2, 3, 1-5, 1-10-, 1-20, or more conservative mutations).

In some embodiments, the ketoreductase comprises an amino acid sequencethat corresponds to SEQ ID NO:2, or is at least about 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:2, and comprises oneor more of the following characteristics:

X⁶ is a polar or acidic amino acid residue;

X²⁷ is an aliphatic or basic amino acid residue;

X³¹ is a hydroxyl containing or aliphatic amino acid residue;

X³⁶ is a small amino acid residue;

X⁴⁸ is a basic amino acid residue;

X⁶³ is a hydrophobic or aromatic amino acid;

X⁶⁵ is a hydroxyl containing or small amino acid residue;

X⁸⁶ is an aliphatic or hydrophobic amino acid residue;

X¹²⁵ is an aliphatic or aromatic amino acid residue;

X¹⁵² is a basic amino acid residue;

X¹⁶⁰ is a hydroxyl containing or small amino acid residue;

X¹⁶⁵ is a polar or acidic amino acid residue;

X¹⁹⁴ is an aromatic amino acid or polar hydrophilic amino acid residue

X²¹⁴ is a polar hydrophilic amino acid residue;

X²¹⁸ is a hydroxyl containing amino acid residue;

X²³⁴ is a polar hydrophilic amino acid residue;

X²⁴⁸ is an aliphatic amino acid residue;

X²⁵⁰ is a basic amino acid residue;

X²⁶³ is a acidic or polar amino acid residue;

X²⁹⁰ is an acidic amino acid residue;

X²⁹⁶ is an aliphatic amino acid residue

X²⁹⁷ is an aromatic amino acid residue;

X³⁰¹ is an aliphatic or hydroxyl containing or acidic or hydrophilic orbasic amino acid residue; and

X³⁰⁷ is an aromatic, a polar or a hydroxyl containing amino acidresidue. In one embodiment, the ketoreductase comprises all of the abovecharacteristics. In one embodiment, the ketoreductase comprises at leastone characteristic such that at least one residue is mutated as comparedto SEQ ID NO:2. In one embodiment, the ketoreductase comprises at leastone characteristic such that at least one residue is non-conservativelymutated as compared to SEQ ID NO:2.

In some embodiments, the ketoreductase enzyme comprises an amino acidsequence that corresponds to SEQ ID NO:2, or is at least about 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:2, andcomprises one or more of the following characteristics:

X⁶ is a polar or acidic amino acid residue;

X²⁷ is an aliphatic or basic amino acid residue;

X³¹ is a hydroxyl containing or aliphatic amino acid residue;

X³⁶ is a small amino acid residue;

X⁴⁸ is a basic amino acid residue;

X⁶³ is an aromatic amino acid;

X⁶⁵ is a hydroxyl containing or small amino acid residue;

X⁸⁶ is an aliphatic or hydrophobic amino acid residue;

X¹²⁵ is an aliphatic or aromatic amino acid residue;

X¹⁵² is a basic amino acid residue;

X¹⁶⁰ is a hydroxyl containing or small amino acid residue;

X¹⁶⁵ is a polar or acidic amino acid residue;

X¹⁹⁴ is an aromatic amino acid or polar hydrophilic amino acid residue

X²¹⁴ is a polar hydrophilic amino acid residue;

X²¹⁸ is a hydroxyl containing amino acid residue;

X²³⁴ is a polar hydrophilic amino acid residue;

X²⁴⁸ is an aliphatic amino acid residue;

X²⁵⁰ is a basic amino acid residue;

X²⁶³ is a acidic or polar amino acid residue;

X²⁹⁰ is an acidic amino acid residue;

X²⁹⁶ is an aliphatic amino acid residue

X²⁹⁷ is an aromatic amino acid residue;

X³⁰¹ is an aliphatic or hydroxyl containing or acidic or hydrophilic orbasic amino acid residue; and

X³⁰⁷ is an aromatic, a polar or a hydroxyl containing amino acidresidue. In one embodiment, the ketoreductase comprises all of the abovecharacteristics. In one embodiment, the ketoreductase comprises at leastone characteristic such that at least one residue is mutated as comparedto SEQ ID NO:2. In one embodiment, the ketoreductase comprises at leastone characteristic such that at least one residue is non-conservativelymutated as compared to SEQ ID NO:2.

In some embodiments, the ketoreductase comprises a ketoreductase havingan improved activity as compared to a ketoreductase of SEQ ID NO:2, andthat has an amino acid sequence that corresponds to the wild-typesequence ketoreductase of FIG. 3 (SEQ ID NO:2) and includes one or moremutations selected from: R27A, R27K, N31S, A36V, Y48H, I63W, R65G, R65S,L86I, F125L, K152R, A160T, Y194C, A218T, V248I, K250R, E290D, L296V,Y297W, Y297F, L301K, L301R, L301A, L301S, L301E, L301P, L301Q, L301V,L301T, L301Y, L301D, Y307H, Y307N, and Y307S. In some embodiments, suchimproved engineered ketoreductases comprise an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:2, with the proviso that the amino acid sequencecomprises at least one or more of the above listed mutations (in someembodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more of the mutations).

In some embodiments, the engineered ketoreductase comprises aketoreductase with an improved enzyme stability having an amino acidsequence that corresponds to the wild-type sequence of FIG. 3 (SEQ IDNO:2) and includes one or more mutations selected from: E44D, I46L,I55V, I56V, A60T, I63V, L76M, G103S, L106I, D114G, L121I, Q144R, S148T,N158H, Q223H, I237G, I237N, I237R, S239T, and V253A.

In some embodiments, the engineered ketoreductase comprises aketoreductase with a combination of improved enzyme activity andimproved enzyme stability having a amino acid sequence that correspondsto the wild type sequence of FIG. 3 (SEQ ID NO:2) and that includes oneor more mutations selected from: Q6E, N31P, A160S, Q165E, Q214H, Q214R,E234Q, Q263E. In one embodiment, the ketoreductase comprises all ofthese mutations.

In some embodiments, the engineered ketoreductase comprises aketoreductase with a combination of improved enzyme activity and/orimproved enzyme stability having an amino acid sequence that correspondsto the wild type sequence of FIG. 3 (SEQ ID NO:2) and includes one ormore mutations selected from: N40Y, V43L, K51E, I58V, E62Q, A73P, K79R,K152Q, K169E, F185I, Q189H, G192D, H200L, D201G, K215E, K215M, K216R,D221V, S233C, I237D, K250E, I265V, L287R, K304R, Q310R, K311E, V312S,and V312E.

Specific exemplary embodiments of such engineered ketoreductases includeSEQ ID NOS: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, and366.

In some embodiments, the engineered ketoreductase enzyme has an aminoacid sequence that is at least about 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% identical to an amino acid sequence listed in Table 1.

In some embodiments, the ketoreductase comprises an amino acid sequencethat is at least about 95, 96, 97, 98, or 99% identical to SEQ ID NO:316or 318. In one embodiment, the ketoreductase comprises the amino acidsequence of SEQ ID NO:316 or 318, but with one or more conservativemutations (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) at residuesthat have not been mutated as compared to SEQ ID NO:2.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductases described herein orpolynucleotides that hybridize to such polynucleotides under highlystringent conditions. The polynucleotide can include promoters and otherregulatory elements useful for expression of the encoded engineeredketoreductase, and can utilize codons optimized for specific desiredexpression systems.

In still another aspect, the present disclosure provides host cellscomprising the polynucleotides and/or expression vectors describedherein. The host cells may be Saccharomyces cerevisiae, which theengineered ketoreductase enzyme was derived from, or they may be adifferent organism. The host cells can be used for the expression andisolation of the engineered ketoreductase enzymes described herein, or,alternatively, they can be used in the various methods described furtherbelow.

The ketoreductase enzymes can be used to reduce hydroxy oxo esterenantiomers of structural formula (Ia) to their corresponding syndihydroxys in high yield and with a high degree of stereoselectivity.

In one aspect, the ketoreductase enzymes are used in purified orisolated form. In another aspect, extracts from cells expressing theenzymes are used.

Thus, in another aspect, the present disclosure provides methods ofproducing a syn dihydroxy ester according to structural formula (IIa):

wherein X and R¹ are as previously defined. The methods generallycomprise selectively reducing a hydroxy oxo ester enantiomer accordingto structural formula (Ia):

wherein X and R¹ are as defined for structural formula (IIa), using anengineered ketoreductase enzyme described herein. The method may becarried out using whole cells that express the engineered ketoreductaseenzyme, such as the host cells described herein, extracts or lysatesfrom such cells, or purified engineered ketoreductase enzymes.

As will be appreciated by skilled artisans, the reduction reactionillustrated above generally requires a cofactor, which is normally NADHor NADPH, and a system for regenerating the cofactor, for exampleglucose and glucose dehydrogenase. Since cells generally provide suchcofactors and cofactor regeneration systems, in embodiments employingwhole cells cell extracts or cell lysates, the addition of suchcofactors and cofactor regeneration systems may be unnecessary. Inembodiments employing purified engineered ketoreductase enzyme(s), suchcofactors and optionally such cofactor regeneration systems willtypically be added to the reaction medium along with the hydroxy oxoester substrate and the ketoreductase enzyme(s). Like the engineeredketoreductase enzyme, any enzyme(s) comprising the cofactor regenerationsystem can be supplied to the reaction mixture in the form of extractsor lysates of such cells, or as purified enzyme(s). In embodimentsemploying cell extracts or cell lysates, the cells used to generate theextracts or lysates can be engineered to express the enzyme(s)comprising the cofactor regeneration systems alone, or together with theengineered ketoreductase enzyme. In embodiments employing whole cells,the cells can be engineered to express the enzyme(s) comprising thecofactor regeneration systems and the engineered ketoreductase enzymetogether.

Whether carrying out the method with whole cells, cell extracts orpurified ketoreductase enzymes, a single ketoreductase enzyme may beused or, alternatively, mixtures of two or more ketoreductase enzymesmay be used.

The ketoreductase enzymes described herein are highly specific for theenantiomer of structural formula (Ia) and catalyze the above-describedreduction reaction in high yield and with a high degree ofstereoselectivity. Depending upon the particular engineeredketoreductase enzyme used, yields in the range of about 80 to 99.9% witha diastereomeric excess (“d.e.”) of ≧99.5%, can be readily achieved. Forexample, it has been discovered that engineered ketoreductase enzymes ofthe present disclosure can be used to stereoselectively reduce the5-hydroxyoxo ester enantiomer of structural formula (Ia), supra, to thecorresponding syn dihydroxy of structural formula (IIa) with yields inthe range of 90 to 99.9% and with a degree of stereoselectivity of≧99.7%.

In many embodiments, diastereoselectivities of ≧99.8%, 99.9%, 99.91%,99.92%, 99.93%, 99.94%, 99.5%, 99.96%, 99.97%, 99.98% and even 99.99%d.e. can be achieved. Indeed, as will be discussed in more detail belowin connection with FIGS. 6 and 7, a crude, unpurified reduction productobtained from a starting substrate that was >99% pure in the enantiomerof structural formula (Ia) using the engineered ketoreductase enzyme ofSEQ ID NO:316 was >99.99% pure in the syn diastereomer of structuralformula (IIa).

Because the engineered ketoreductase enzymes described herein are sohighly stereoselective, the resultant syn dihydroxy ester of structuralformula (IIa) can be recovered in substantially stereochemically pureform without the need to chirally separate it from the correspondinganti or any other diastereomer. Indeed, owing to the high yield and highstereoselectivity of the reaction, if the hydroxy oxo ester substrate ofstructural formula (I) is substantially pure in the enantiomer ofstructural formula (Ia), the specific syn dihydroxy ester diastereomerof structural formula (IIa) can be recovered from the reaction medium asa substantially pure or pure diastereomer without purification.

The chiral dihydroxy ester of structural formula (IIa) can be used as astarting material to synthesize cholesterol-lowering statins, includingbut not limited to, atorvastatin, rosuvastatin, and pitavastatin. A keystep in many of these methods is protecting the hydroxyl groups of thesyn dihydroxy ester of structural formula (IIa), yielding a protectedsyn dihydroxy ester according to structural formula (IIIa):

-   -   wherein X and R¹ are as previously defined for structural        formula (IIa) and each R² is a protecting group or,        alternatively, the two R² groups can be taken together to form a        substituted or unsubstituted alkylene bridge. In some        embodiments, the protected syn dihydroxy ester is a compound        according to structural formula (IVa):

-   -   wherein X and R¹ are as previously defined for structural        formula (IIa) and R³ and R⁴ are each, independently of one        another, selected from hydrogen, (C1-C12) alkyl, (C6-C10) aryl        and (C7-C12) arylalkyl, or alternatively, R³ and R⁴ taken        together form a (C4-C10) cycloalkyl or heterocycloalkyl. In a        specific embodiment, R³ and R⁴ are each methyl.

In some embodiments, the protected syn dihydroxy ester is a compoundaccording to structural formula (Va):

wherein X and R¹ are as previously defined for structural formula (IIa),and R⁵ is selected from (C1-C12) alkyl, (C6-C10) aryl and (C7-C12)arylalkyl. In some embodiments, R⁵ is phenyl.

Owing to their stereoselectivity, the engineered ketoreductase enzymesdescribed herein can be used to synthesize stereoisomerically purepreparations of protected syn dihydroxy esters according to structuralformula (IIIa), (IVa) and (Va). The methods generally compriseenantioselectively reducing a hydroxyoxoester enantiomer according tostructural formula (Ia), supra, using an engineered ketoreductase enzymeas described herein to yield a first reaction product comprising a syndihydroxy ester according to structural formula (IIa), supra, andprotecting the hydroxyl groups of the syn dihydroxy ester to yield theprotected syn dihydroxy ester of structural formula (IIIa). Owing to thehigh stereoselectivity of the engineered ketoreductase enzymes, theprotected syn dihydroxy ester of structural formula (IIIa) can berecovered as a substantially stereochemically pure or pure diastereomer,i.e., substantially free of the corresponding anti diastereomer and anyother diastereomers, without chiral separation.

The syn dihydroxy esters and/or protected syn dihydroxy esters can beused as reactants in known methods to synthesize cholesterol loweringstatins. Exemplary synthetic routes for preparing exemplary statins areillustrated in FIGS. 8, 9, and 10. Thus, in another aspect, the presentdisclosure provides methods of producing substantiallystereoisomerically pure preparations of statins, such as, for example,atorvastatin, rosuvastatin, and pitavastatin. The methods generallycomprises: (i) reducing a substantially enantiomerically pure hydroxyoxo ester enantiomer according to structural formula (Ia):

wherein R¹ and X are as previously defined, with an engineeredketoreductase enzyme described herein to yield a first reaction productcomprising a substantially diastereomerically pure syn dihydroxy esteraccording to structural formula (IIa):

wherein X and R¹ are as defined for structural formula (Ia); and (ii)thereafter using the syn dihydroxy ester of structural formula (IIa) tosynthesize a statin. Since the engineered ketoreductases describedherein catalyze the reduction of step (i) with a high degree ofenantioselectivity, the syn dihydroxy ester of structural formula (IIa)can be used to synthesize statins having a high degree of stereoisomericpurity using reactions carried out at nearly ambient temperature andwithout the need for chiral separations. Indeed, the high degree ofstereoselectivity of the reduction reaction allows preparation ofstatins having a % d.e. that is within about 90 to 99.99% of the % e.e.of the hydroxy oxo ester starting substrate, readily and economically inhigh yield.

6. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the role of ketoreductases and halohydrindehalogenases in converting an alkyl 4-halo-3-oxobutyrate to variousintermediates useful in the synthesis of statins.

FIG. 2 illustrates various routes for synthesizing t-butyl(6R)-(2-cyanomethyl)-2,2 dimethyl-1,3-dioxane-(4R)-acetate from at-butyl-3,5-dihydroxy-6-halohexanoate.

FIG. 3 provides the wild-type sequence of the ketoreductase encoded bythe Ydl124wp gene (Genbank Acc. No.: NP_(—)010159.1; GI: 6320079) fromSaccharomyces cerevisiae (SEQ ID NO:2).

FIG. 4 provides the amino acid sequence of the ketoreductase fromSaccharomyces cerevisiae with the residues marked that can be modifiedto generate engineered polypeptides with improved ketoreductaseproperties.

FIG. 5 provides an illustration of plasmid pCK110900 in which clonedketoreductase genes are operatively linked to the lac promoter undercontrol of the lad repressor for expressing engineered ketoreductaseenzymes.

FIG. 6 shows LC/MS/MS chromatograms of t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate samples of Example 17 (a sampleprepared by an embodiment of the methods described herein) (upper panel)and a sample prepared from commercial crystalline t-butyl(6R)-(2-cyanoethyl)-2,2 dimethyl-1,3-dioxane-(4R)-acetate (lower panel).

FIG. 7 shows GC/MS chromatograms of t-butyl (6R)-(2-cyanoethyl)-2,2dimethyl-1,3 dioxane-(4R)-acetate samples of Example 23 (a sampleprepared by the method of the invention) and a commercial crystallinesample.

FIG. 8 illustrates a synthetic scheme for rosuvastatin; R represents Hor a suitable protecting group.

FIG. 9 illustrates a synthetic scheme for pitavastatin; R represents Hor a suitable protecting group.

FIG. 10 illustrates a synthetic scheme for atorvastatin; R represents Hor a suitable protecting group.

7. DETAILED DESCRIPTION 7.1 Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. “Lower alkyl”refers to an alkyl group containing from 1 to 6 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C1-C6means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies be on the same carbonatom, the nomenclature “alkylidene” is used. A “lower alkyldiyl” is analkyldiyl group containing 1 to 6 carbon atoms. In some embodiments thealkyldiyl groups are saturated acyclic alkanyldiyl groups in which theradical centers are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenes, defined infra).

“Alkylene” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkylene is indicatedin square brackets. Typical alkylene groups include, but are not limitedto, methylene (methano); ethylenes such as ethano, etheno, ethyno;propylenes such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno,etc.; butylenes such as butano, but[1]eno, but[2]eno, buta[1,3]dieno,but[1]yno, but[2]yno, buta[1,3]diyno, etc.; and the like. Where specificlevels of saturation are intended, the nomenclature alkano, alkenoand/or alkyno is used. In some embodiments, the alkylene group is(C1-C6) or (C1-C3) alkylene. In some embodiments, the alkylene group isa straight-chain saturated alkano group, e.g., methano, ethano, propano,butano, and the like.

“Cycloalkyl” by itself or as part of another substituent refers to acyclic version of an “alkyl” group. Typical cycloalkyl groups include,but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyland cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl;cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In some embodiments, the aryl group is(C6-C10) Specific examples are phenyl and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specifically saturated orunsaturated alkyl moieties are intended, the nomenclature arylalkanyl,arylalkenyl and/or arylalkynyl is used. In some embodiments, thearylalkyl group is (C7-C12) arylalkyl. In a specific embodiment thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C2) andthe aryl moiety is (C6-C10).

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide that is capable of enantioselectively reducing the 3-oxogroup of a 5-hydroxy-3-oxohexanoate ester enantiomer to yield thecorresponding syn 3,-5-dihydroxyhexanoate ester. The polypeptidetypically utilizes the cofactor reduced nicotinamide adeninedinucleotide (NADH) or reduced nicotinamide adenine dinucleotidephosphate (NADPH) as the reducing agent. Ketoreductases as used hereininclude naturally occurring (wild type) ketoreductases as well asnon-naturally occurring engineered polypeptides generated by humanmanipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, by the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WisconsinSoftware Package), or by visual inspection (see generally, CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 andAltschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas, the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a “comparison window” to identify and compare local regions ofsequence similarity.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly reported in the art (typically as a percentage) asthe enantiomeric excess calculated therefrom according to the formula[major enantiomer−minor enantiomer]/[major enantiomer+minor enantiomer].Where the stereoisomers are diastereoisomers, the stereoselectivity isreferred to as diastereoselectivity, the fraction (typically reported asa percentage) of one diastereomer in the sum with others. In the contextof the present disclosure, diastereoselectivity refers to the fraction(typically reported as a percentage) of the hydroxy oxo ester ofstructural formula (Ia) that gets converted into the syn dihydroxy esterof structural formula IIa, as opposed to the anti dihydroxy ester offormula IIb. It may also be reported (typically as a percentage) as thediastereomeric excess calculated therefrom according to the formula [synIIa−anti IIb]/[syn IIa+anti IIb].

7.2 Ketoreductase Enzymes

The present disclosure provides engineered ketoreductase (“KRED”)enzymes that are capable of enantioselectively reducing the 3-oxo groupof a 5-hydroxy-3-oxohexanoate ester enantiomer to produce thecorresponding syn 3,5-dihydroxyhexanoate ester, and have at least oneimproved property when compared with the naturally-occurring, wild-typeKRED enzyme obtained from Saccharomyces cerevisiae. As noted above, thestereoisomerically pure syn 3,5-dihydroxyhexanoate esters produced bythe ketoreductase can serve as intermediates for the synthesis ofseveral cholesterol lowering statins. Improvements in various enzymeproperties can facilitate use of these engineered ketoreductases inlarge scale production of this intermediate compound. Any number ofimprovements in enzymatic properties would be useful for the describedapplication, for example, enzymatic activity, thermal stability, andreduced cofactor requirement.

Ketoreductase enzymes having improved properties can be obtained bymutating the genetic material encoding the ketoreductase enzyme ofSaccharomyces cerevisiae and identifying polynucleotides that expressengineered enzymes with a desired property. These non-naturallyoccurring ketoreductases can be generated by various well-knowntechniques, such as in vitro mutagenesis or directed evolution. In someembodiments, directed evolution is an attractive method for generatingengineered enzymes because of the relative ease of generating mutationsthroughout the whole of the gene coding for the polypeptide, as well asproviding the ability to take previously mutated polynucleotides andsubjecting them to additional cycles of mutagenesis and/or recombinationto obtain further improvements in a selected enzyme property. Subjectingthe whole gene to mutagenesis can reduce the bias that may result fromrestricting the changes to a limited region of the gene. It can alsoenhance generation of enzymes affected in different enzyme propertiessince distantly spaced parts of the enzyme may play a role in variousaspects of enzyme function. Mutagenesis and directed evolutiontechniques useful for the purposes herein are amply described in theliterature: Ling, et al., 1997, “Approaches to DNA mutagenesis: anoverview,” Anal. Biochem. 254(2):157-78; Dale et al., 1996,“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod,” Methods Mol. Biol. 57:369-74; Smith, 1985, “In vitromutagenesis,” Ann. Rev. Genet. 19:423-462; Botstein et al., 1985,“Strategies and applications of in vitro mutagenesis,” Science229:1193-1201; Carter, 1986, “Site-directed mutagenesis,” Biochem. J.237:1-7; Kramer et al., 1984, “Point Mismatch Repair,” Cell, 38:879-887;Wells et al., 1985, “Cassette mutagenesis: an efficient method forgeneration of multiple mutations at defined sites,” Gene 34:315-323;Minshull et al., 1999, “Protein evolution by molecular breeding,” CurrOpin Chem Biol 3:284-290; Christians et al., 1999, “Directed evolutionof thymidine kinase for AZT phosphorylation using DNA family shuffling,”Nature Biotech 17:259-264; Crameri et al., 1998, “DNA shuffling of afamily of genes from diverse species accelerates directed evolution,”Nature 391:288-291; Crameri et al., 1997, “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotech15:436-438; Zhang et al., 1997, “Directed evolution of an effectivefructosidase from a galactosidase by DNA shuffling and screening,” ProcNatl Acad Sci USA 94:45-4-4509; Crameri et al., 1996, “Improved greenfluorescent protein by molecular evolution using DNA shuffling,” NatureBiotech 14:315-319; Stemmer, 1994, “Rapid evolution of a protein invitro by DNA shuffling,” Nature 370:389-391; Stemmer, 1994, “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution,” Proc Natl Acad Sci USA 91:10747-10751; WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767and U.S. Pat. No. 6,537,746. All publications are incorporated herein byreference.

The naturally occurring polynucleotide encoding the naturally occurringketoreductase of Saccharomyces cerevisiae can be obtained from theisolated polynucleotide known to encode the ketoreductase activity(Genbank accession no. NP_(—)010159 GI: 6320079). Alternatively, apolynucleotide encoding the naturally occurring ketoreductases can besynthesized by polynucleotide synthesis methodologies known in the artbased on the reported polynucleotide sequence of the Saccharomycescerevisiae ketoreductase-encoding gene. In various embodiments, asfurther described below, the naturally occurring polynucleotide encodingthe ketoreductase can be codon optimized to the codons preferred by aspecific host cell used for expression of the enzyme. See e.g., Example1.

The parent or reference polynucleotide encoding the naturally occurringor wild type ketoreductase is subjected to mutagenic processes, forexample random mutagenesis and recombination, to introduce mutationsinto the polynucleotide. The mutated polynucleotide is expressed andtranslated, thereby generating engineered ketoreductase enzymes withmodifications to the polypeptide. As used herein, “modifications”include amino acid substitutions, deletions, and insertions. Any one ora combination of modifications can be introduced into the naturallyoccurring enzymatically active polypeptide to generate engineeredenzymes, which are then screened by various methods to identifypolypeptides, and corresponding polynucleotides, having a desiredimprovement in a specific enzyme property. A polynucleotide encoding anengineered ketoreductase with an improved property can be subjected toadditional rounds of mutagenesis treatments to generate polypeptideswith further improvements in the desired enzyme property.

As used herein, a ketoreductase enzyme that has an “improved enzymeproperty” refers to a ketoreductase enzyme that exhibits an improvementin any enzyme property as compared to a reference ketoreductase enzyme.For the engineered ketoreductase enzymes described herein, thecomparison is generally made to the wild-type Saccharomyces cerevisiaeketoreductase enzyme (SEQ ID NO:2), although in some embodiments, thereference ketoreductase can be another improved engineeredketoreductase. Enzyme properties for which improvement is desirableinclude, but are not limited to, enzymatic activity, thermal stability,pH activity profile, cofactor requirements, refractoriness to inhibitors(e.g., product inhibition), sterospecificity, and stereoselectivity.

As used herein the context of engineered ketoreductase enzymes, “derivedfrom” identifies the originating ketoreductase enzyme, and/or the geneencoding such ketoreductase enzyme, upon which the engineering wasbased. For example, the engineered ketoreductase enzyme of SEQ ID NO:80was obtained by artificially evolving, over multiple generations thegene encoding the Saccharomyces cerevisiae ketoreductase enzyme of SEQID NO:2. Thus, this engineered ketoreductase enzyme is “derived from”the wild-type ketoreductase of SEQ ID NO.: 2.

While not intending to be bound by any theory of operation, it isbelieved that the improvements in enzyme properties arise fromintroduction of modifications into a polypeptide chain that, in effect,perturb the structure-function of the enzyme and/or its interactionswith another molecule (e.g., substrate). Some regions of the polypeptidemay be critical to enzyme activity, for example amino acids involved incatalysis and substrate binding domains, such that small perturbationsto these regions may have significant effects on enzyme function. Someamino acid residues may be at important positions for maintaining thesecondary or tertiary structure of the enzyme, and thus also producenoticeable changes in enzyme properties when modified. On the otherhand, some regions of the polypeptide may have a global structural roleor form part of the enzyme less involved in the critical aspects ofenzyme function (e.g., a peptide loop connecting alpha helices) andproduce a measurable effect on enzyme function only when a large numberof substitutions have accumulated in the region. Thus, in someembodiments, the number of modifications to the naturally occurringpolypeptide that produces an improved ketoreductase property maycomprise one or more amino acids, 2 or more amino acids, 5 or more aminoacids, 10 or more amino acids, or 20 or more amino acids, up to 10% ofthe total number of amino acids, up to 20% of the total number of aminoacids, or up to 30% of the total number of amino acids of the referenceenzyme sequence.

Because of the nature of the technique, gene evolution approaches, suchas DNA mutagenesis and recombination, biases the modification towardsamino acid substitutions. Thus, in various embodiments, the improvementsin enzyme properties can arise from substitutions at one amino acidresidue, 2 or more amino acid residues, 5 or more amino acid residues,10 or more amino acid residue, 15 or more amino acid residues, 20 ormore amino acid residues, up to 10% of the total number of amino acids,up to 20% of the total number of amino acids, or up to 30% of the totalnumber of amino acids making up the reference enzyme sequence.

As will be apparent to the skilled artisan, an analysis of amino acidsubstitutions for a large number of engineered ketoreductase with animproved enzyme property shows that the substitutions recur at certainamino acid residues in the ketoreductase polypeptide as well as a biastowards certain types of amino acids. This recurrence of substitutionsat certain defined positions within the polypeptide may reflect theeffect of amino acid residue on that particular enzyme property and itsretention by the continued imposition of selection for the particularimproved enzyme property. The recurrence of substitutions as well as thebias of the types of substituted amino acids can be grouped to describethe types of substitutions allowable for generating the improved enzymeproperty. Typically, genetically encoded amino acids can be grouped intothe following classes based on their side chain properties:

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-His (H), L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) is classified above asa basic residue, as its side chain includes a heteroaromatic ring, itmay also be classified as an aromatic residue.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

The amino acid L-Cys (C) is unusual in that it can form disulfidebridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized as apolar hydrophilic amino acid, notwithstanding the generalclassifications defined above.

The amino acid Gly (G) is also unusual in that it bears no side chain onits α-carbon and, as a consequence, contributes only a peptide bond to aparticular peptide sequence. Moreover, owing to the lack of a sidechain, it is the only genetically-encoded amino acid having an achiralα-carbon. Although Gly (G) exhibits a hydrophobicity of 0.48 accordingto the normalized consensus scale of Eisenberg (Eisenberg et al., 1984,supra), for purposes of the present invention, Gly is categorized as analiphatic amino acid or residue.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include Gly,L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) andL-Asp (D).

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Indeed, the delineated categoryof small amino acids includes amino acids from all of the otherdelineated categories except the aromatic category. Thus, amino acidshaving side chains exhibiting two or more physico-chemical propertiescan be included in multiple categories. As a specific example, aminoacid side chains having heteroaromatic moieties that include ionizableheteroatoms, such as His, may exhibit both aromatic properties and basicproperties, and can therefore be included in both the aromatic and basiccategories. The appropriate classification of any amino acid or residuewill be apparent to those of skill in the art, especially in light ofthe detailed disclosure provided herein.

In some embodiments, the substitution in the engineered ketoreductasemay be a conservative substitution. The term “conservative amino acidsubstitutions” refer to the interchangeability of residues havingsimilar side chains, and thus typically involves substitution of theamino acid in the polypeptide with amino acids within the same orsimilar defined class of amino acids. By way of example and notlimitation, an amino acid with an aliphatic side chain may besubstituted with another aliphatic amino acid, e.g., alanine, valine,leucine, isoleucine, and methionine; an amino acid with hydroxyl sidechain is substituted with another amino acid with a hydroxyl side chain,e.g., serine and threonine; an amino acids having aromatic side chainsis substituted with another amino acid having an aromatic side chain,e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acidwith a basic side chain is substituted with another amino acid with abasis side chain, e.g., lysine, arginine, and histidine; an amino acidwith an acidic side chain is substituted with another amino acid with anacidic side chain, e.g., aspartic acid or glutamic acid; and ahydrophobic or hydrophilic amino acid is replaced with anotherhydrophobic or hydrophilic amino acid, respectively.

In some embodiments, the substitution in the engineered ketoreductasemay be a conservative substitution. The term “conservative amino acidsubstitutions” refer to the interchangeability of residues havingsimilar side chains, and thus typically involves substitution of theamino acid in the polypeptide with amino acids within the same orsimilar defined class of amino acids. By way of example and notlimitation, an amino acid with an aliphatic side chain may besubstituted with another aliphatic amino acid, e.g., alanine, valine,leucine, isoleucine, and methionine; an amino acid with hydroxyl sidechain is substituted with another amino acid with a hydroxyl side chain,e.g., serine and threonine; an amino acids having aromatic side chainsis substituted with another amino acid having an aromatic side chain,e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acidwith a basic side chain is substituted with another amino acid with abasis side chain, e.g., lysine, arginine, and histidine; an amino acidwith an acidic side chain is substituted with another amino acid with anacidic side chain, e.g., aspartic acid or glutamic acid; and ahydrophobic or hydrophilic amino acid is replaced with anotherhydrophobic or hydrophilic amino acid, respectively.

In various embodiments, the substitutions for generating an improvedketoreductase can comprise conservative substitutions, non-conservativesubstitutions, as well as combinations of conservative andnon-conservative substitutions.

In some embodiments, the improved engineered ketoreductase enzymescomprise deletions of the naturally occurring ketoreductase polypeptidesas well as deletions of other improved ketoreductase polypeptides. Theterm “deletion” refers to modification to the polypeptide by removal ofone or more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered ketoreductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

In other embodiments, the improved engineered ketoreductase enzymescomprise insertions of one or more amino acids to the naturallyoccurring ketoreductase polypeptide as well as insertions of one or moreamino acids to other improved ketoreductase polypeptides. Insertions canbe in the internal portions of the polypeptide, or to the carboxy oramino terminus Insertions as used herein include fusion proteins as isknown in the art. The insertion can be a contiguous segment of aminoacids or separated by one or more of the amino acids in the naturallyoccurring polypeptide.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered ketoreductaseenzyme can be targeted to a specific property of the enzyme. Thus, insome embodiments, the improved property of the engineered ketoreductasepolypeptides is an increased enzymatic activity, typically representedby an increase in specific activity (e.g., product produced/time/weightprotein) as compared to the reference ketoreductase enzyme (i.e., thenaturally occurring ketoreductase). Exemplary methods to determineenzyme activity are provided in the Examples. Any property relating toenzyme activity may be affected, including the classical enzymeproperties of K_(m), V_(max) or k_(cat), changes of which can lead toincreased enzymatic activity. Improvements in enzyme activity can befrom about 1.5 times the enzymatic activity of the correspondingwild-type ketoreductase enzyme, to as much as 2 times. 5 times, 10times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200times, 250 times, 300 times, or more enzymatic activity than thenaturally occurring ketoreductase of SEQ ID NO:2. In specificembodiments, the engineered ketoreductase enzyme exhibits improvedenzymatic activity in the range of 1.5 to 50 times, 1.5 to 100 times,1.5 to 150 times, 1.5 to 200 times, 1.5 to 250 times, or 1.5 to 300times greater than that of the wild-type ketoreductase enzyme. It isunderstood by the skilled artisan that the activity of any enzyme isdiffusion limited such that the catalytic turnover rate cannot exceedthe diffusion rate of the substrate, including any required cofactors.The theoretical maximum of the diffusion limit, or k_(cat)/K_(m), isgenerally about 10⁸ to 10⁹ (M⁻¹ s⁻¹). Hence, any improvements in theenzyme activity of the ketoreductase will have an upper limit related tothe diffusion rate of the substrates acted on by the ketoreductaseenzyme. Ketoreductase activity can be measured by any one of standardassays used for measuring ketoreductase, such as a decrease inabsorbance or fluorescence of NADPH due to its oxidation with theconcomitant reduction of a ketone to the corresponding alcohol, or byproduct produced in a coupled assay. Comparisons of enzyme activitiesare made using a defined preparation of enzyme and a defined assay undera set condition, as further described in detail herein. Generally, whenlysates are compared, the numbers of cells and the amount of proteinassayed are determined as well as use of identical expression systemsand identical host cells to minimize variations in amount of enzymeproduced by the host cells and present in the lysates.

In some embodiments, the engineered ketoreductase with improved enzymeactivity comprise engineered polypeptides derived from Saccharomycescerevisiae ketoreductase of SEQ ID NO:2. In some embodiments, theengineered ketoreductase comprises an amino acid sequence that is atleast about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQID NO:2. Amino acid residues that can be modified to generate engineeredpolypeptides with improved enzyme activity are shown in FIG. 4 in whichX^(n) represents the modified amino acid residue. In some embodiments,one or more of the naturally occurring amino acid residue of SEQ ID NO:2can be substituted as follows:

X⁶ is a polar or acidic amino acid residue;

X²⁷ is an aliphatic or basic amino acid residue;

X³¹ is a hydroxyl containing or aliphatic amino acid residue;

X³⁶ is a small amino acid residue;

X⁴⁸ is a basic amino acid residue;

X⁶³ is a hydrophobic or aromatic amino acid;

X⁶⁵ is a hydroxyl containing or small amino acid residue;

X⁸⁶ is an aliphatic or hydrophobic amino acid residue;

X¹²⁵ is an aliphatic or aromatic amino acid residue;

X¹⁵² is a basic amino acid residue;

X¹⁶⁰ is a hydroxyl containing or small amino acid residue;

X¹⁶⁵ is a polar or acidic amino acid residue;

X¹⁹⁴ is an aromatic amino acid or polar hydrophilic amino acid residue

X²¹⁴ is a polar hydrophilic amino acid residue;

X²¹⁸ is a hydroxyl containing amino acid residue;

X²³⁴ is a polar hydrophilic amino acid residue;

X²⁴⁸ is an aliphatic amino acid residue;

X²⁵⁰ is a basic amino acid residue;

X²⁶³ is a acidic or polar amino acid residue;

X²⁹⁰ is an acidic amino acid residue;

X²⁹⁶ is an aliphatic amino acid residue

X²⁹⁷ is an aromatic amino acid residue;

X³⁰¹ is an aliphatic, hydroxyl containing, acidic, hydrophilic, or basicamino acid residue; and/or

X³⁰⁷ is an aromatic, a polar or a hydroxyl containing amino acidresidue. In some embodiments, the ketoreductase has all of these residuecharacteristics.

In some embodiments, the ketoreductase has one or more of these residuecharacteristics. In some embodiments, at least one of these residuepositions does not include a substitution that is a conservativemutation with respect to SEQ ID NO:2. In some embodiments, X⁶³ is anaromatic residue. In some embodiments, X⁶³ is tryptophan. In someembodiments, residues at other positions can be conservatively mutatedwith respect to SEQ ID NO:2.

In some embodiments, the amino acid residues of the ketoreductase of SEQID NO:2 can be substituted with one or more specific amino acids asfollows:

X⁶ is E;

X²⁷ is A or K;

X³¹ is S or P;

X³⁶ is V;

X⁴⁸ is H;

X⁶³ is W;

X⁶⁵ is G or S;

X⁸⁶ is I;

X¹²⁵ is L;

X¹⁵² is R;

X¹⁶⁰ is T or S;

X¹⁶⁵ is E;

X¹⁹⁴ is C;

X²¹⁴ is R or H;

X²¹⁸ is T;

X²³⁴ is Q;

X²⁴⁸ is I;

X²⁵⁰ is R;

X²⁶³ is E;

X²⁹⁰ is V or D;

X²⁹⁶ is I;

X²⁹⁷ is F or W;

X³⁰¹ is R, K, A, V, S, T, Y, D, E, P, or Q; and/or

X³⁰⁷ is H, N or S.

In some embodiments, the engineered ketoreductases with improved enzymeactivity have substitutions in a subset of the amino acid residuesindicated above. Thus, in some embodiments the amino acid substitutionscan occur in one or more of the amino acid positions: X²⁷, X³¹, X³⁶,X⁴⁸, X⁶³, X⁶⁵, X⁸⁶, X¹²⁵, X¹⁵², X¹⁶⁰, X¹⁹⁴, X²¹⁸, X²⁴⁸, X²⁵⁰, X²⁹⁰,X²⁹⁶, X²⁹⁷, X³⁰¹, and X³⁰⁷, wherein the amino acid residues forsubstitution are selected as indicated above.

In some embodiments, the engineered ketoreductase comprises one or moremutations selected from: R27A, R27K, N31S, A36V, Y48H, I63W, R65G, R65S,L86I, F125L, K152R, A160T, Y194C, A218T, V248I, K250R, E290D, L296V,Y297W, Y297F, L301K, L301R, L301A, L301S, L301E, L301P, L301Q, L301V,L301T, L301Y, L301D, Y307H, Y307N, and Y307S.

It is also shown herein that the various amino acid residue positionswhose substitutions result in a ketoreductase with improved propertiesare found to cluster in certain segments of the ketoreductase enzyme,which allows targeted modifications of the segments. Accordingly, insome embodiments, selected segments of the ketoreductase polypeptide maybe subject to modification. For the reference sequence of SEQ ID NO:2,the segments affecting ketoreductase enzymatic activity include, amongothers, those represented by amino acid residues 27 to 36, 40 to 65, 103to 125, 144 to 169, 233 to 265, and/or 287 to 312. One or more of theamino acid residues within each segment or combination of segments maybe modified. In some embodiments, the corresponding amino acid sequencesand positions for substitutions comprise:

X²⁷WYKX³¹EETDX³⁶ X⁴⁰SLX⁴³X⁴⁴QX⁴⁶VX⁴⁸ALX⁵¹LPGX⁵⁵X⁵⁶HX⁵⁸DX⁶⁰AX⁶²X⁶³YX⁶⁵X¹⁰³LDX¹⁰⁶ALKK MGTX¹¹⁴YVDLYLX¹²¹HSPX¹²⁵X¹⁴⁴LYKX¹⁴⁸GKAX¹⁵²NIGVSX¹⁵⁸F X¹⁶⁰VEDLX¹⁶⁵RILX¹⁶⁹X²³³X²³⁴KYX²³⁷KX²³⁹EAQIILRWX²⁴⁸TX²⁵⁰RGX²⁵³LPVTTSS KPX²⁶³RX²⁶⁵; andX²⁹⁰HEPLRX²⁹⁶X²⁹⁷WNKX³⁰¹YGKYNX³⁰⁷AAX³¹⁰X³¹¹X³¹²wherein X^(n) indicates positions of the amino acid substitutions.

In some embodiments, the engineered ketoreductase enzyme with improvedenzymatic activity has an amino acid sequence selected from the aminoacid sequences recited in Table 1:

TABLE 1 temperature Nucleic Acid Protein for stability SEQ ID NO. SEQ IDNO. Residue substitutions Activity test (° C.) Stability 15 16 L106I; *37 + 17 18 G112R; A160T; * 37 + 19 20 E44D; Y307S; * 37 + 21 22 I22M;S148T; * 37 + 23 24 E290D; * 37 + 25 26 R27A; * 40 — 27 28 V253A;Y307N; * 40 — 29 30 R27A; Y48H; * 40 — 31 32 A60T; * 40 + 33 34 D114G; *40 + 35 36 F185L; * 40 — 37 38 N31S; * 40 — 39 40 L296V; * 40 — 41 42E78K; A160T; * 40 — 43 44 N40Y; S233C; * 40 — 45 46 Q214H; * 40 — 47 48V248I; * 40 — 49 50 E62Q; F185I; * 40 — 51 52 A60T; * 40 + 53 54 R27A;Y48H; A160T; * 40 — 55 56 I22M; A101V; G112R; A160T; * 40 — Y307S; 57 58R27A; Y307S; * 40 — 59 60 R27K; K51E; S148T; I237N; * 40 + 61 62 A60T;D102G; I237N; * 40 + 63 64 I237N; * 40 + 65 66 R27A; Y48H; A160T; * 40 —67 68 A60T; I237N; * 40 + 69 70 I22M; A60T; I237N; * 40 + 7{grave over( )} 72 R27A; A160T; K261R; * 40 — 73 74 I22M; I237N; V253A; * 40 + 7576 I22M; E44D; I237N; E290Q; * 40 + 77 78 A160T; E290D; * 40 — 79 80I22M; I237N; E290D; * 40 + 81 82 G112R; A160T; Y307S; * 40 — 83 84 I22M;T77A; G112R; A160T; * 40 — 85 86 I22M; I237N; * 40 + 87 88 N31S;I237N; * 40 + 89 90 A60T; I237T; * 40 + 91 92 A160T; Y307S; * 40 — 93 94K147R; A160T; I237N; Y307S; * 40 + 95 96 R27K; A160T; I237N; * 40 + 9798 R27K; K79R; A160T; * 40 — 99 100 R27A; G112R; A160T; I237N; * 40 —101 102 A60T; K195R; I237N; * 40 + 103 104 A160T; * 40 — 105 106 I63V;E78K; A160T; Q214R; * 40 — 107 108 K51E; Y194C; * 40 — 109 110 N31S;I237T; * 40 — 111 112 R27K; A160T; Q214R; I237N; ** 40 + 113 114 R27K;A160T; S233G; I237N; ** 40 — 115 116 R27K; D114N; A160T; I237N; ** 40 —V248I; 117 118 R27K; A160T; I237N; V253A; * 40 + 119 120 R27K; V43F;A160T; I237N; ** 40 — 121 122 R27K; A160T; V229I; I237N; * 40 + 123 124R27K; F125L; A160T; I237N; ** 40 — 125 126 R27K; L121I; A160T; I237N; *40 + 127 128 R27K; A160T; I237N; K261M; ** 40 — 129 130 A160S; I237R; **40 + 131 132 A160S; I237G; ** 40 + 133 134 A160S; I237Q; ** 40 + 135 136R27K; L76M; A160T; I237N; * 40 + 137 138 R27K; S148R; A160T; I237N; **40 — 139 140 R27K; A160T; I237N; S239T; * 40 + 141 142 R27K; A160T;S222R; Q223H; * 40 + I237N; 143 144 R27K; V47L; A160T; D221V; * 40 +I237N; 145 146 R27K; A160T; I237N; L296Q; ** 40 — 147 148 R27K; A160S;Q214R; I237N; ** 43 + 149 150 R27K; Y48H; A160T; Q214R; *** 43 — I237N;151 152 R27K; K51E; I63V; L121I; ** 43 + A160T; Q214R; I237G; 153 154R27K; N31S; L121I; A160T; *** 43 + Q214R; K215M; I237N; 155 156 R27A;N31S; A160T; H200L; *** 43 — Q214R; K215E; I237N; 157 158 R27A; N31S;A160T; Q214R; *** 43 — I237N; V312E; 159 160 R27K; N31S; I58V; A160T;*** 43 — Q214R; I237N; I265V; 161 162 R27A; N31S; Y48H; A160T; *** 43 —Q214R; I237N; 163 164 R27K; A160T; G192D; Q214R; *** 43 — I237N; K250E;165 166 R27K; I63V; L121I; F125L; ** 43 + A160T; Q214R; I237N; 167 168R27K; K152R; A160T; Q214R; *** 43 — I237N; 169 170 R27A; N31S; A160T;Q214R; *** 43 — I237N; 171 172 R27K; G103S; A160T; Q214R; ** 43 + I237N;173 174 R27K; A60T; I84V; A160T; * 43 + Q214R; I237N; 175 176 R27K;A160T; Q214R; I237R; *** 43 — Y307S; 177 178 R27A; N31S; F125L; A160T;*** 43 — K169R; Q214R; I237N; 179 180 R27K; Q144R; A160T; Q214R; ** 43 +I237N; 181 182 R27K; L121I; A160T; Q214R; *** 43 + I237D; 183 184 R27K;N31S; A160T; Q214R; *** 43 — I237N; Y307S; 185 186 R27K; L121I; A160T;Y194C; *** 43 + Q214R; I237N; 187 188 R27K; N31S; E78K; AS60T; *** 43 —Q214R; T217A; S222G; I237N; 189 190 R27K; L121I; A160T; Q214R; ** 43 +I237G; 191 192 R27K; A160T; Q214R; Q223H; ** 43 + I237N; 193 194 R27K;E78K; A160T; Q214R; *** 43 — I237N; Y307S; 195 196 R27A; N31S; LI21I;A160T; *** 43 + Q214R; I237N; 197 198 R27K; L121I; A160T; Y194C; ***43 + Q214R; I237N; 199 200 R27K; N31S; L121I; A160T; *** 43 + Q214R;I237N; 201 202 R27K; N31S; A160T; Q214R; *** 43 — I237G; 203 204 I19T;R27K; N31S; A160T; *** 43 — Q214R; I237G; 205 206 R27K; I63V; A160T;Q214R; ** 43 + I237N; 207 208 R27K; N31S; K51E; A160T; *** 43 — Q214R;I237N; K304R; 209 210 R27K; N31S; L121I; A160T; *** 43 + Y194C; Q214R;I237R; 211 212 R27K; A160T; G192D; Q214R; *** 43 — I237N; Y307S; 213 214R27A; N31S; A160T; Y194C; *** 43 — Q214R; I237R; 215 216 R27K; E78K;LI21I; A160T; ** 43 + Q214R; K216R; I237G; 217 218 R27K; A160T; Q214R;I237G; ** 43 + 219 220 R27K; I56V; A160T; Q214R; ** 43 + I237N; 221 222R27K; N31S; I63V; A160T; ** 43 + Q214R; I237N; 223 224 R27K; A160T;Q214R; I237N; *** 43 — K250R; 225 226 R27K; V130G; A160T; Q214R; *** 43— I237N; 227 228 R27K; L121I; A160T; Q214R; ** 43 + I237N; 229 230 R27K;A160T; Q189H; Q214R; ** 43 + I237N; L287R; 231 232 R27K; A73P; A160T;D201G; *** 43 — Q214R; I237G; 233 234 R27K; A160T; Q214R; I237N; *** 43— Y307H; 235 236 R27K; N31S; L121I; A160T; *** 43 + Y194C; Q214R; I237N;237 238 R27K; N31S; L121I; A160S; *** 43 + Y194C; Q214R; I237N; 239 240R27K; N31S; S148T; A160T; *** 43 — Q214R; I237N; Y307S; 241 242 R27K;L121I; A160S; Y194C; *** 43 + Q214R; I237N; 243 244 R27K; N31S; E78K;A160T; *** 43 — Q214R; I237G; Y307S; 245 246 R27K; N31S; L121I; S127T;*** 43 + A160T; Y194C; Q214R; I237N; 247 248 R27A; N31S; L121I; A160S;**** 43 + Y194C; Q214R; I237N; 249 250 R27K; N31S; L121I; S148T; ****43 + A160T; Y194C; Q214R; I237N; 251 252 R27K; N31S; L121I; A160T; ****43 + Y194C; Q214R; I237G; 253 254 R27K; N31S; L106I; L121I; **** 43 +A160S; Y194C; Q214R; I237N; 255 256 R27A; N31S; L121I; A160S; **** 43 +Q214R; I237N; 257 258 R27K; N31S; L121I; S148T; **** 43 + A160S; Y194C;Q214R; I237N; 259 260 R27A; N31S; F125L; A160T; **** 45 — Y194C; Q214R;I237N; 261 262 R27A; N31S; F125L; A160T; **** 45 — K169R; Q214R; I237N;263 264 R27A; N31S; F125L; A160T; **** 45 — Q214R; I237R; 265 266 R27K;N31S; L121I; F125L; **** 45 + A160T; Y194C; Q214R; K215M; I237N; 267 268R27A; N31S; S148T; A160T; **** 45 — K169R; Q214R; I237N; 269 270 I21T;R27A; N31S; L121I; **** 45 — F125L; A160T; Q214R; I237N; 271 272 R27A;N31S; F125L; A160T; **** 45 — Y194C; Q214R; I237R; 273 274 R27A; N31S;F125L; A160T; **** 45 — Q214R; I237N; 275 276 R27A; N31S; L121I; F125L;**** 45 — A160T; Y194C; Q214R; I237N; 277 278 R27A; N31P; L121I; F125L;**** 48 — A160T; Y194C; Q214R; I237N; 279 280 I19V; R27A; N31S; E44D;K72R; **** 48 — D105E; L121I; F125L; A160T; F185L; Y194C; Q214R; I237N;I243V; E290D; 281 282 Q6E; R27A; N31S; S41T; L121I; **** 48 + F125L;A160T; Y194C; E199Q; Q214R; I237N; L271I; 283 284 Q6E; R27A; N31S; S41T;I46L; **** 48 + I55V; L121I; F125L; A160T; Y194C; E199Q; Q214R; I237N;285 286 Q6E; R27A; N31S; S41T; I46L; **** 48 + F125L; Q144E; A160T;Y194C; E199Q; Q214R; I237N; S239T; L271I; 287 288 Q6E; R27A; N31S; I46L;L121I; **** 48 + F125L; A160T; Y194C; Q214R; I237N; S239T; I244L; 289290 Q6E; R27A; N31S; L121I; F125L; **** 48 + A160T; Y194C; Q214R; I237N;S239T; 291 292 Q6E; R27A; N31S; S41T; L121I; **** 48 + F125L; A160T;Y194C; Q214R; I237N; L271I; 293 294 Q6E; R27A; N31S; L121I; F125L; ****48 + A160T; Y194C; Q214R; E234Q; I237N; S239T; 295 296 Q6E; R27A; N31S;I58L; F125L; **** 48 — A160T; Y194C; E199Q; Q214R; I237N; I244L; 297 298Q6E; R27A; N31S; I46L; L121I; **** 48 + F125L; A160T; Y194C; E199Q;Q214R; I237N; S239T; I244L; 299 300 Q6E; R27A; N31S; I58L; L86I; ****48 + L121I; F125L; A160T; Y194C; Q214R; I237N; S239T; I244L; 301 302Q6E; R27A; N31S; I46L; L121I; **** 48 + F125L; A160T; Y194C; Q214R;I237N; S239T; 303 304 Q6E; R27A; N31S; L121I; F125L; **** 48 + A160T;Y194C; Q214R; I237N; L271I; 305 306 Q6E; R27A; N31S; I46L; I58L; **** 48— L121I; F125L; Q144E; A160T; Y194C; Q214R; I237N; A309T; 307 308 Q6E;R27A; N31S; L121I; F125L; **** 48 — Q144E; A160T; Q165E; Y194C; E199Q;Q214R; I237N; S239T; I244L; Y305H; 309 310 Q6E; R27A; N31S; S41T; I46L;**** 48 + L121I; F125L; A160T; Y194C; Q214R; I237N; 311 312 Q6E; R27A;N31S; S41T; I46L; **** 48 + L121I; F125L; A160T; Y194C; Q214R; I237N;313 314 Q6E; R27A; N31P; I46L; L86I; **** 50 + L121I; F125L; A160T;Y194C; Q214R; I237N; S239T; 315 316 R27A; N31P; I63W; L121I; **** 50 —F125L; A160T; Y194C; Q214R; I237N; 317 318 R27A; N31P; I63W; L121I; ****50 — F125L; A160T; Y194C; Q214R; I237N; 319 320 R27A; N31P; L121I;F125L; **** 50 — A160T; Y194C; Q214R; I237N; Y297W; 321 322 R27A; N31P;L121I; F125L; **** 50 — A160T; Y194C; Q214R; I237N; L301R; 323 324 R27A;N31P; L121I; F125L; **** 50 — A160T; Y194C; Q214R; I237N; L301K; 325 326R27A; N31P; I63W; L121I; **** 50 — F125L; A160T; Y194C; Q214R; I237N;Y297W; 327 328 R27A; N31P; I63W; L121I; **** 50 — F125L; A160T; Y194C;Q214R; I237N; Y297W; L301K; 329 330 Q6E; R27A; N31P; I46L; I63W; ****50 + L86I; L121I; F125L; A160T; Y194C; Q214R; I237N; S239T; 331 332R27A; N31P; I63W; L121I; **** ND ND F125L; A160T; Y194C; Q214R; I237N;Y297W; L301A; 333 334 R27A; N31P; I63W; L121I; **** ND ND F125L; A160T;Y194C; Q214R; I237N; Y297W; L301S; 335 336 R27A; N31P; I63W; L121I; ****ND ND F125L; A160T; Y194C; Q214R; I237N; Y297W; L301E; 337 338 R27A;N31P; I63W; L121I; **** ND ND F125L; A160T; Y194C; Q214R; I237N; Y297W;L301P; 339 340 R27A; N31P; I63W; L121I; **** ND ND F125L; A160T; Y194C;Q214R; I237N; Y297W; L301Q; 341 342 R27A; N31P; I63W; L121I; **** 50 —F125L; A160T; Y194C; Q214R; I237N; L301E; 343 344 R27A; N31P; S41G;I63W; L121I; **** 50 — F125L; A160T; Y194C; Q214R; I237N; L301A; 345 346R27A; N31P; I63W; L121I; **** 50 — F125L; A160T; Y194C; Q214R; I237N;Y297F; L301E; 347 348 R27A; N31P; I63W; R65G; **** 50 — L121I; F125L;A160T; Y194C; Q214R; I237N; Y297W; L301Q; 349 350 R27A; N31P; I63W;R65S; L121I; **** 50 — F125L; A160T; Y194C; Q214R; I237N; Y297W; L301Q;351 352 R27A; N31P; A36V; I63W; **** 50 — L121I; F125L; A160T; Y194C;Q214R; I237N; Y297W; L301Q; 353 354 R27A; N31P; I63W; L121I; **** 50 —F125L; A160T; Y194C; Q214R; I237N; Y297W; L301Q; Q310R; K311E; V312S;*313V; 355 356 R27A; N31P; I63W; L121I; **** 50 — F125L; A160T; Y194C;Q214R; A218T; I237N; Y297W; L301Q; 357 358 R27A; N31P; I63W; L121I; ****50 — F125L; A160T; Y194C; Q214R; I237N; Y297W; L301Q; Q310P; K311E;V312S; *313V; 359 360 Q6E; R27A; N31P; I46L; I63W; **** ND ND L86I;L121I; F125L; A160T; Y194C; Q214R; I237N; S239T; L301E; 361 362 Q6E;R27A; N31P; I46L; L86I; **** ND ND L121I; F125L; A160T; Y194C; Q214R;I237N; S239T; Y297W; 363 364 Q6E; R27A; N31P; I46L; I63W; **** ND NDL86I; L121I; F125L; A160T; Y194C; Q214R; I237N; S239T; Y297W; 365 366Q6E; R27A; N31P; I46L; I63W; **** ND ND L86I; L121I; F125L; A160T;Y194C; Q214R; I237N; S239T; Y297W; L301Q; * = 150-500% enzymaticactivity of SEQ ID NO: 2 ** = 500-900% enzymatic activity of SEQ ID NO:2 *** = 900-1600% enzymatic activity of SEQ ID NO: 2 **** = greater than1600% enzymatic activity of SEQ ID NO: 2 + = variant retains residualactivity after heat treatment at indicated temperature for 16 hrs. ND =Not Determined

In other embodiments, the engineered ketoreductase comprises aketoreductase with improved thermal stability as compared to thenaturally occurring ketoreductase from which the engineered enzymes arederived. Thermally stable enzymes allow longer incubations withsubstrate, such as in conditions of high substrate concentrations usedin large scale production, and also minimize the amount of enzymeneeded. An enzyme that has “improved thermal stability” refers to anenzyme that displays increased resistance to inactivation when exposedto a set temperature or set of temperatures in defined assay conditionsas compared to the resistance to inactivation of a reference enzyme.

A number of methods are available for measuring enzyme thermalstability. Enzymes may be treated for a defined time period at varioustemperatures under a standard set of assay conditions (e.g., ionicstrength, pH, protein concentration, etc.). Half-inactivationtemperatures, i.e., temperatures at which 50% inactivation occurs afterheat treatment for the defined time are used to compare the thermalstabilities of the enzymes. Another method of determining thermalstability is to treat the enzyme at a set temperature and measure theamount of enzyme activity remaining. The amount of enzyme activityremaining is compared to the activity remaining of the naturallyoccurring enzyme prepared under similar or identical conditions. Usefultemperatures for analysis of thermal stability are temperatureencompassing the reaction temperature for used for carrying out thereaction, for example as in large scale reactions, and/or temperaturesat which the engineered enzymes have been selected for improved thermalstability. For example, the engineered enzymes can have improved thermalstability as compared to a reference enzyme (e.g., a naturally occurringenzyme) at temperatures of about 30° C. or above, about 35° C. or above,40° C. or above, 45° C. or above, 50° C. or above, 55° C. or above, toabout 65° C. In some embodiments, the enzyme can have an improvedthermal stability in which the engineered enzyme retains, as compared toa reference enzyme, about 10% or more activity, about 15% or moreactivity, about 20% or more activity, about 30% or more activity, about50% or more activity, about 70% or more activity, about 90% or moreactivity. Under the conditions of treatment, the times at which enzymeactivity shows enhanced thermal stability can include, about 5 hrs ormore, about 6 hrs or more, about 12 hrs or more, about 18 hrs or more,about 24 hrs or more, about 36 hrs or more, about 48 hrs or more, about72 hrs or more. Substrate may or may not be present during thermaltreatment, although presence of substrate is known to stabilize enzymestructures in some instances.

In other embodiments, the thermal stability of an enzyme can be measuredby determining protein folding stability, such as by detecting proteinunfolding with calorimetry or circular dichroism. For example, todetermine the thermal stability of the engineered and naturallyoccurring ketoreductase, changes in CD profile at various temperaturescan be used. An Arrhenius plot of the unfolding rate constants of theenzymes is generated by use of temperature jumps (e.g., from atemperature where an enzyme is active to a temperature where the enzymeis inactive) and measuring changes in protein structure. The first-orderrate constants, activation enthalpies, and activation free energiesbetween the stable and transition state of the unfolding reaction at aninactivating temperature may be compared. Typically, the activation freeenergy increases with more thermally stable enzymes. Other methods fordetermining thermal stability will be apparent to the skilled artisan.

In some embodiments, the improved thermal stability is separable fromother properties of the ketoreductase enzymes, while in otherembodiments, the thermal stability is also observed with anotherimproved property. In some embodiments, the ketoreductases compriseengineered ketoreductases with improved thermal stability is derivedfrom the Saccharomyces cerevisiae ketoreductase of SEQ ID NO:2. In someembodiments, the engineered ketoreductase with improved thermalstability retains about 20% or more of enzyme activity followingtreatment at temperature ranging from 37 to 50° C. for 16 hrs at pH 7.0.By comparison, the naturally occurring ketoreductase of SEQ ID NO:2retains only 5-10% of its activity after incubation at 37° C. for 16 hrsat pH 7.0 and retains essentially no activity when incubated at highertemperatures under the same conditions.

In some embodiments, the engineered ketoreductases with improved thermalstability comprises an amino acid sequence corresponding to SEQ ID NO:2and includes one or more of the following substitutions:

X⁶ is a polar or acidic amino acid residue;

X³¹ is a hydroxyl containing or aliphatic amino acid residue;

X⁴⁴ is an acidic amino acid residue;

X⁴⁶ is an aliphatic amino acid residue

X⁵⁵ is an aliphatic amino acid residue;

X⁵⁶ is an aliphatic amino acid;

X⁶⁰ is an hydroxyl containing or small amino acid;

X⁶³ is a hydrophobic or aromatic amino acid;

X⁷⁶ is a amino acid residue M;

X¹⁰³ is a hydroxyl containing amino acid residue;

X¹⁰⁶ is an aliphatic amino acid residue;

X¹¹⁴ is a small amino acid residue;

X¹²¹ is a hydrophobic or aliphatic amino acid residue;

X¹⁴⁴ is a hydrophilic or basic amino acid residue

X¹⁴⁸ is a hydroxyl containing amino acid residue

X¹⁵⁸ is an aromatic amino acid residue

X¹⁶⁰ is a hydroxyl containing or small amino acid residue;

X¹⁶⁵ is a polar or acidic amino acid residue;

X¹⁶⁹ is a aliphatic or basic amino acid residue;

X¹⁸⁵ is an aromatic or aliphatic amino acid residue;

X¹⁹⁴ is an aromatic amino acid or polar hydrophilic amino acid residue

X¹⁹⁹ is a polar amino acid residue;

X²¹⁴ is an aromatic or basic amino acid residue;

X²²³ is an aromatic amino acid residue;

X²³⁴ is a polar hydrophilic amino acid residue;

X²³⁷ is a hydrophilic or small amino acid residue;

X²³⁹ is a hydroxyl containing amino acid residue;

X²⁵³ is an aliphatic amino acid residue; and/or

X²⁶³ is a acidic or polar amino acid residue.

In some embodiments, X⁶³ is an aromatic residue. In some embodiments,X⁶³ is tryptophan. In some embodiments, the ketoreductase comprises oneor more conservative mutations at other positions than those listedabove.

In some embodiments, the engineered ketoreductases with improved thermalstability comprises an amino acid sequence corresponding to SEQ ID NO:2and includes one or more of the following substitutions:

X⁶ is E;

X³¹ is P;

X⁴⁴ is D;

X⁴⁶ is L;

X⁵⁵ is V;

X⁵⁶ is V;

X⁶⁰ is T;

X⁶³ is V;

X⁷⁶ is M;

X¹⁰³ is S;

X¹⁰⁶ is I;

X¹¹⁴ is G;

X¹²¹ is I or M;

X¹⁴⁴ is R;

X¹⁴⁸ is T;

X¹⁵⁸ is H;

X¹⁶⁰ is S;

X¹⁶⁵ is E;

X²¹⁴ is R or H;

X²²³ is H, R or G;

X²³⁴ is Q;

X²³⁷ is N, G, R or Q;

X²³⁹ is T; and/or

X²⁵³ is A.

In some embodiments, the engineered ketoreductase with improved thermalstability have substitutions in a subset of the amino acid residuesindicated above. Thus, in some embodiments the amino acid substitutionscan occur at one or more of the amino acid positions X⁴⁴, X⁴⁶, X⁵⁵, X⁵⁶,X⁶⁰, X⁶³, X⁷⁶, X¹⁰³, X¹⁰⁶, X¹¹⁴, X¹²¹, X¹⁴⁴, X¹⁴⁸, X¹⁵⁸, X²²³, X²³⁷,X²³⁹, and X²⁵³, wherein the amino acid residues for substitution areselected as indicated above.

In some embodiments, the engineered ketoreductase with improved thermalstability comprises one or more of the following substitutions ascompared to SEQ ID NO:2: E44D, I46L, I55V, I56V, A60T, I63V, L76M,G103S, L106I, D114G, L121I, Q144R, S148T, N158H, Q223H, I237G, I237N,I237R, S239T, and V253A.

As observed for the engineered ketoreductases with improved enzymaticactivity, various amino acid residue positions whose substitutionsresult in an engineered ketoreductase with improved thermal stabilityare also found to cluster in certain segments of the ketoreductase ofSEQ ID NO:2, which allows targeted modifications. Substitutionsresulting in improved thermal stability overlap with those that occur inengineered enzymes with improved enzymatic activity, although some donot. Accordingly, in some embodiments, selected segments of theketoreductase polypeptide may be subject to modification to generateengineered ketoreductases with improved thermal stability. For thereference sequence of SEQ ID NO:2, the segments affecting ketoreductaseenzymatic activity include, among others, those represented by aminoacid residues 27 to 36, 40 to 65, 103 to 125, 144 to 169, 233 to 265,and 287 to 312. In some embodiments, the segments affectingketoreductase enzymatic activity include, among others, thoserepresented by amino acid residues 27-32, 60-65, 90-93, 120-125,157-159, 208-211, and 293-306. One or more of the amino acid residues ineach segment or combination of segments may be modified.

In some embodiments, the corresponding amino acid sequences andpositions for substitutions comprise:

X²⁷WYKX³¹EETDX³⁶ X⁴⁰SLX⁴³X⁴⁴VX⁴⁸ALX⁵¹LPGX⁵⁵X⁵⁶HX⁵⁸DX⁶⁰AX⁶²X⁶³YX⁶⁵X¹⁰³LDX¹⁰⁶ALKK MGTX¹¹⁴YVDLYLX¹²¹HSPX¹²⁵X¹⁴⁴LYKX¹⁴⁸GKAX¹⁵²NIGVSX¹⁵⁸F X¹⁶⁰VEDLX¹⁶⁵RILX¹⁶⁹X²³³X²³⁴KYX²³⁷KX²³⁹EAQIILRWX²⁴⁸TX²⁵⁰ RGX²⁵³LPVTTSS KPX²⁶³RX²⁶⁵; andX²⁹⁰HEPLRX²⁹⁶X²⁹⁷WNKX³⁰¹YGKYNX³⁰⁷AAX³¹⁰X³¹¹X³¹²

wherein X^(n) indicates positions of the amino acid substitutions. Bothconservative and non-conservative substations can be made in thesegments as indicated.

In some embodiments, the improved property can be a combination ofproperties, such as improved enzymatic activity and improved thermalstability. In some embodiments, the engineered ketoreductase comprises aketoreductase with a combination of improved enzyme activity andimproved enzyme stability having a amino acid sequence that correspondsto the wild type sequence of the ketoreductase of SEQ ID NO:2 andincludes one or more mutations selected from: Q6E, N31P, A160S, Q165E,Q214H, Q214R, E234Q, Q263E.

In some embodiments, the engineered ketoreductase comprises aketoreductase with a combination of improved enzyme activity and/orstability having an amino acid sequence that corresponds to the wildtype sequence of the ketoreductase SEQ ID NO:2 and includes one or moremutations selected from: N40Y, V43L, K51E, I58V, E62Q, A73P, K79R,K152Q, K169E, F185I, Q189H, G192D, H200L, D201G, K215E, K215M, K216R,D221V, S233C, I237D, K250E, I265V, L287R, K304R, Q310R, K311E, V312S,V312E.

It is to be understood that the engineered ketoreductases are notlimited to those identified directly by mutagenesis or other geneevolution techniques, but also include variants or analogs of theimproved engineered ketoreductase polypeptides. The term “variant” or“analog” as used herein refers to polypeptides which are comprised of asegment having ketoreductase activity, with or without retention of theimproved property, and has substantial identity to a portion of theengineered ketoreductase. In some embodiments, analog polypeptidescomprise a conservative amino acid substitution, or addition or deletionof one or more amino acid residues with respect to the engineeredsequence. Analogs typically are at least an enzymatically activefragment and typically as long as the full-length naturally-occurringreference polypeptide of SEQ ID NO:2 (e.g., 312 amino acid residues).

In some embodiments, the variant or analog of the engineeredketoreductase polypeptides can comprise an enzymatically activeketoreductase polypeptide that has about 70% or more amino acididentity, about 80% or more amino acid identity, about 90% or more aminoacid identity, about 95% or more amino acid identity, about 97% or moreamino acid identity, about 98% or more amino acid identity, or about 99%or more amino acid identity to a reference engineered ketoreductase oran enzymatically active fragment of an engineered ketoreductase.

In some embodiments, the reference engineered ketoreductase is selectedfrom the amino acid sequences recited in Table 1. An exemplary referencesequence of an improved ketoreductase is SEQ ID NO:314. In someembodiments, the variant or analog of the engineered ketoreductasepolypeptides can comprise an enzymatically active ketoreductasepolypeptide that has about 95.5% or more amino acid identity, about 97%or more amino acid identity, about 98% or more amino acid identity, orabout 99% or more amino acid identity to SEQ ID NO:314, or anenzymatically active fragment of SEQ ID NO:314.

Another exemplary reference sequence of an improved ketoreductase is SEQID NO:316. In some embodiments, the variant or analog of the engineeredketoreductase polypeptides can comprise an enzymatically activeketoreductase polypeptide that has about 95.5% or more amino acididentity, about 97% or more amino acid identity, about 98% or more aminoacid identity, or about 99% or more amino acid identity to SEQ ID NO:316or an enzymatically active fragment of SEQ ID NO:316.

Another exemplary reference sequence of an improved ketoreductase is SEQID NO:318. In some embodiments, the variant or analog of the engineeredketoreductase polypeptides can comprise an enzymatically activeketoreductase polypeptide that has about 95.5% or more amino acididentity, about 97% or more amino acid identity, about 98% or more aminoacid identity, or about 99% or more amino acid identity to SEQ ID NO:318or an enzymatically active fragment of SEQ ID NO:318.

Another exemplary reference sequence of an improved ketorreductase isSEQ ID NO:376. In some embodiments, the variant or analog of theengineered ketoreductase polypeptides can comprise an enzymaticallyactive ketoreductase polypeptide that has about 95.5% or more amino acididentity, about 97% or more amino acid identity, about 98% or more aminoacid identity, or about 99% or more amino acid identity to SEQ ID NO:376or an enzymatically active fragment of SEQ ID NO:376.

In some embodiments, segments of the improved ketoreductase polypeptidescan be deleted to generate polypeptide fragments. The term “fragment” asused herein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion, but where the remaining amino acid sequenceis identical to the corresponding positions in the sequence. Fragmentscan be at least 14 amino acids long, at least 20 amino acids long, atleast 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%,and 99% of the full-length naturally-occurring ketoreductase polypeptideof SEQ ID NO:2.

The improved ketoreductase enzymes may be present within a cell, presentin the cellular medium, or prepared in various forms, such as lysates orisolated preparations. As such, in some embodiments, the improvedketoreductase enzyme can be an isolated polypeptide. The term “isolatedpolypeptide” refers to a polypeptide which is substantially separatedfrom other contaminants that naturally accompany it, e.g., protein,lipids, and polynucleotides. The term embraces polypeptides which havebeen removed or purified from their naturally-occurring environment orexpression system (e.g., host cell or in vitro synthesis).

In some embodiments, the isolated improved ketoreductases polypeptide isa substantially pure polypeptide composition. The term “substantiallypure polypeptide” refers to a composition in which the polypeptidespecies is the predominant species present (i.e., on a molar or weightbasis it is more abundant than any other individual macromolecularspecies in the composition), and is generally a substantially purifiedcomposition when the object species comprises at least about 50 percentof the macromolecular species present by mole or % weight. Generally, asubstantially pure ketoreductase composition will comprise about 60% ormore, about 70% or more, about 80% or more, about 90% or more, about 95%or more, and about 98% or more of all macromolecular species by mole or% weight present in the composition. In some embodiments, the objectspecies is purified to essential homogeneity (i.e., contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

7.3 Polynucleotides Encoding Engineered Ketoreductases

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductase enzymes. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered ketoreductase canbe introduced into appropriate host cells to express the correspondingketoreductase polypeptide. The term “heterologous” polynucleotide refersto any polynucleotide that is introduced into a host cell by laboratorytechniques, and includes polynucleotides that are removed from a hostcell, subjected to laboratory manipulation, and then reintroduced into ahost cell.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved ketoreductaseenzymes disclosed herein. Thus, having identified a particular aminoacid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in Table 1.

In some embodiments, the polynucleotides encoding the ketoreductasesenzymes may be codon optimized for optimal production from the hostorganism selected for expression. The term “codon optimized” refers tochanges in the codons of the polynucleotide encoding a protein to thosepreferentially used in a particular organism such that the encodedprotein is efficiently expressed in the organism of interest. Althoughthe genetic code is degenerate in that most amino acids are representedby several codons, called “synonyms” or “synonymous” codons, it is wellknown that codon usage by particular organisms is nonrandom and biasedtowards particular codon triplets. This codon usage bias may be higherin reference to a given gene, genes of common function or ancestralorigin, highly expressed proteins versus low copy number proteins, andthe aggregate protein coding regions of an organism's genome.

The terms “preferred,” “optimal,” or “high codon usage bias” codonsrefers interchangeably to codons that are used at higher frequency inthe protein coding regions than other codons that code for the sameamino acid. The preferred codons may be determined in relation to codonusage in a single gene, a set of genes of common function or origin,highly expressed genes, the codon frequency in the aggregate proteincoding regions of the whole organism, codon frequency in the aggregateprotein coding regions of related organisms, or combinations thereof.Codons whose frequency increases with the level of gene expression aretypically optimal codons for expression.

A variety of methods are known for determining the codon frequency(e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (see GCG CodonPreference,Genetics Computer Group Wisconsin Package; CodonW, John Peden,University of Nottingham; McInerney, J. O, 1998, Bioinformatics14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright,F., 1990, Gene 87:23-29).

Codon usage tables are available for a growing list of organisms (seefor example, Wada et al., 1992, Nucleic Acids Res. 20:2111-2118;Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra;Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt,et al. Eds., ASM Press, Washington D.C., p. 2047-2066. The data sourcefor obtaining codon usage may rely on any available nucleotide sequencecapable of coding for a protein. These data sets include nucleic acidsequences actually known to encode expressed proteins (e.g., completeprotein coding sequences-CDS), expressed sequence tags (ESTS), orpredicted coding regions of genomic sequences (see for example, Mount,D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. By way of example, the polynucleotide of SEQ ID NO: 1 has beencodon optimized for expression in E. coli, but otherwise encodes thenaturally occurring ketoreductase of Saccharomyces cerevisiae.

In certain embodiments, all codons need not be replaced to optimize thecodon usage of the ketoreductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the ketoreductase enzymes may contain preferredcodons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codonpositions of the full length coding region.

In the embodiments, the polynucleotides encoding the engineeredketoreductases are derived from Saccharomyces cerevisiae ydl gene. Insome embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NOS: 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, and 365.

These polynucleotides encode the corresponding polypeptides representedby the amino acid sequences listed in Table 1, which were derived bysubjecting the E. coli codon optimized Saccharomyces ydl gene todirected gene evolution techniques described herein.

In other embodiments, the polynucleotides comprise polynucleotides thathave about 80% or more sequence identity, about 85% or more sequenceidentity, about 90% or more sequence identity, about 95% or moresequence identity, about 98% or more sequence identity, or 99% or moresequence identity at the nucleotide level to a reference polynucleotideencoding an engineered ketoreductase with improved enzymatic propertiesor a fragment of an engineered ketoreductase with improved properties.In some embodiments, the reference polynucleotide is selected frompolynucleotide sequences represented by SEQ ID NOS: 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, and 365.

In some embodiments, the polynucleotide encodes an enzymatically activeketoreductase and hybridizes under defined conditions, such asmoderately stringent or highly stringent conditions, to the complementof a sequence encoding an engineered ketoreductase enzyme of the presentdisclosure. The phrase “stringent hybridization” is used herein to referto conditions under which nucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of ion strength, temperature, G/C content, andthe presence of chaotropic agents. The T_(m) values for polynucleotidescan be calculated using known methods for predicting meltingtemperatures (see, e.g., Baldino et al., Methods Enzymology 168:761-777;Bolton et al., 1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer etal., 1986, Proc. Natl. Acad. Sci. USA 83:8893-8897; Freier et al., 1986,Proc. Natl. Acad. Sci USA 83:9373-9377; Kierzek et al., Biochemistry25:7840-7846; Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412(erratum, 1991, Nucleic Acids Res 19:698); Sambrook et al., supra);Suggs et al., 1981, In Developmental Biology Using Purified Genes (Brownet al., eds.), pp. 683-693, Academic Press; and Wetmur, 1991, Crit RevBiochem Mol Biol 26:227-259. All publications incorporate herein byreference).

Generally, hybridization reactions are performed under conditions oflower stringency, followed by washes of varying but higher stringency.Typically, reference to “hybridization stringency” relates to suchwashing conditions. The term “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% identity, preferably about 75% identity, about85% identity to the target DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C.

The term “high stringency hybridization” refers generally to conditionsthat are about 10° C. or less from the thermal melting temperature T_(m)as determined under the solution condition for a defined polynucleotidesequence. In some embodiments, a high stringency condition refers toconditions that permit hybridization of only those nucleic acidsequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if ahybrid is not stable in 0.018M NaCl at 65° C., it will not be stableunder high stringency conditions, as contemplated herein). Highstringency conditions can be provided, for example, by hybridization inconditions equivalent to 50% formamide, 5×Denhart's solution, 5×SSPE,0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

The term “control sequence” is defined herein to include all components,which are necessary or advantageous for the expression of a polypeptideof the present disclosure. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polynucleotide and/or polypeptide.

The control sequence may be an appropriate promoter sequence. The“promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of the coding region. The promoter sequencecontains transcriptional control sequences, which mediate the expressionof the polypeptide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promotersare described in “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94; and in Sambrook et al., supra.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the KRED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding animproved ketoreductase polypeptide or a variant thereof, and one or moreexpression regulating regions such as a promoter and a terminator, areplication origin, etc., depending on the type of hosts into which theyare to be introduced. The various nucleic acid and control sequencesdescribed above may be joined together to produce a recombinantexpression vector which may include one or more convenient restrictionsites to allow for insertion or substitution of the nucleic acidsequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell.

Furthermore, the integrational elements may be non-encoding or encodingnucleic acid sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori (as shown in the plasmid of FIG. 5) or the origins of replication ofplasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), orpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,or pAMβ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes it's functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTM™ expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

7.4 Host Cells for Expression of Ketoreductase Polypeptides

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase enzyme in the host cell. Host cells for use inexpressing the KRED polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the ketoreductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110 as described inExample 1. The expression vector was created by operatively linking apolynucleotide encoding an improved ketoreductase into the plasmidpCK110900 operatively linked to the lac promoter under control of thelacI repressor. The expression vector also contained the P15a origin ofreplication and the chloramphenicol resistance gene. Cells containingthe subject polynucleotide in Escherichia coli W3110 were isolated bysubjecting the cells to chloramphenicol selection.

7.5 Methods of Generating Engineered Ketoreductase Polypeptides

To make the improved KRED polynucleotides and polypeptides of thepresent disclosure, the naturally-occurring ketoreductase enzyme thatcatalyzes the reduction reaction is obtained from Saccharomycescerevisiae. In some embodiments, the parent polynucleotide sequence iscodon optimized to enhance expression of the ketoreductase in aspecified host cell. As an illustration, the parental polynucleotidesequence encoding the wild-type KRED polypeptide (ydl) of Saccharomycescerevisiae was constructed from oligonucleotides composed of 42nucleotides based upon the known polypeptide sequence of Saccharomycescerevisiae KRED sequence (ydl) available in Genbank database(NP_(—)010159; GI: 6320079). The parental polynucleotide sequence,designated as SEQ ID NO: 1, was codon optimized for expression in E.coli and the codon-optimized polynucleotide cloned into the SfiI cloningsites of the expression vector, pCK110900 (depicted in FIG. 5), placingthe expression of the ketoreductase gene under the control of the lacpromoter and lad repressor gene. The expression vector also containedthe P15A origin of replication and the chloramphenicol resistance gene.Clones expressing the active ketoreductase in E. coli W3110 wereidentified and the genes sequenced to confirm their identity. Thesequence designated ydlC (SEQ ID NO: 1) was the parent sequence utilizedas the starting point for all experiments and library construction ofengineered ketoreductases evolved from the S. cerevisiae ketoreductase.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods. An exemplary directedevolution technique is mutagenesis and/or DNA shuffling as described inStemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746. Other directed evolution procedures that can be usedinclude, among others, staggered extension process (StEP), in vitrorecombination (Zhao et al., 1998, Nat. Biotechnol. 16:258-261),mutagenic PCR (Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140),and cassette mutagenesis (Black et al., 1996, Proc Natl Acad Sci USA93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD⁺ or NADP⁺. In this reaction,the NADH or NADPH is consumed (oxidized) by the ketoreductase as theketoreductase reduces a ketone substrate to the corresponding hydroxylgroup. The rate of decrease of NADH or NADPH concentration, as measuredby the decrease in absorbance or fluorescence, per unit time indicatesthe relative (enzymatic) activity of the KRED polypeptide in a fixedamount of the lysate (or a lyophilized powder made therefrom). Example11 discloses an exemplary biochemical assay for ketoreductase activity.Where the improved enzyme property desired is thermal stability, enzymeactivity may be measured after subjecting the enzyme preparations to adefined temperature and measuring the amount of enzyme activityremaining after heat treatments, such as in Example 12. Clonescontaining a polynucleotide encoding a ketoreductase are then isolated,sequenced to identify the nucleotide sequence changes (if any), and usedto express the enzyme in a host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Midland Certified Reagent Company, Midland, Tex.,The Great American Gene Company, Ramona, Calif., ExpressGen Inc.Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a compound. The compound may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette Guerin) andCorynebacterium parvum.

An exemplary process for recovering the KRED polypeptides from celllysate for applications in a chemical process is disclosed in Example 2.

7.6 Methods of Using the Engineered Ketoreductase Enzymes and CompoundsPrepared Therewith

The engineered ketoreductase enzymes described herein catalyze thereduction of 5-hydroxy-3-oxohexanoate enantiomers according tostructural formula (Ia):

-   -   wherein X and R¹ are as previously defined, to yield the        corresponding syn 3,5-dihydroxyhexanoate ester compound. As will        be recognized by skilled artisans, the carbon at the 5-position        of the compound of structural formula (I) is chiral. As a        consequence, the compound can exist in two different        enantiomeric forms: the enantiomer of structural formula (Ia)        above and the enantiomer of structural formula (Ib):

Skilled artisans will also recognize that when the oxo group at the3-position is reduced to a hydroxy group, the carbon atom of the3-position in the resultant 3,5-dihydroxy ester is also chiral. Thus,each of the enantiomers of structural formulae (Ia) and (Ib) can yieldtwo different reduction products: a syn diastereomer and antidiastereomer. The syn and anti diastereomers produced by reduction ofenantiomer (Ia) are illustrated below as compounds (IIa) and (IIb),respectively, and those produced by reduction of enantiomer (Ib) areillustrated as compounds (IIc) and (IId), respectively:

The absolute configurations about the 3-carbon are illustrated in theabove structural diagrams. The R— or S— designations for the absoluteconfigurations about the 5-carbon will depend upon the identity of theX. When X is cyano, the 5-carbon is designated R— in structures (Ia),(IIa) and (IIb) and designated S— in structures (Ib), (IIc) and (IId).When X is halo or hydroxy, the 5-carbon is designated S— in structures(Ia), (IIa) and (IIb) and designated R— in structures (Ib), (IIc) and(IId). This is due to the conventions for designating R or S, whichreverse between X=cyano and X=halo or hydroxyl.

In some embodiments, the substrate (Ia) is of high enantiopurity,substantially free of (Ib).

In some embodiments, the reduction reaction is also highlydiastereoselective, producing the syn diastereomer of structural formula(IIa) in ≧99.95% diasteromeric purity, i.e. ≦0.05% of the antidiastereomer of structural formula (IIb) (i.e., ≧.99.9% diastereomericexcess). Many embodiments of the engineered ketoreductases describedherein exhibit even higher stereoselectivities. For example, inreactions of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (structuralformula (Ia), X═CN, R¹=t-butyl) carried out with the engineeredketoreductases referenced in Table 2, diastereoselectivities for6-cyano-(3R,5R)-dihydroxyhexanoate (structural formula (IIa), X═CN,R¹=t-butyl) of ≧99.99% have been observed. Thus, diastereoselectivereductions ranging from ≧99.9%, ≧99.91%, ≧99.92%, ≧99.93%, ≧99.94%,≧99.95%, ≧99.96%, ≧99.97%, ≧99.98% and even ≧99.99% diastereoselectivitycan be readily achieved with the engineered ketoreductase enzymesdescribed herein.

The high degree of diastereoselectivity observed in reduction reactionscatalyzed by the engineered ketoreductases described herein enables theability to produce preparations of the syn 3,5-dihydroxyhexanoate esterof structural formula (IIa) that are substantially diastereomericallypure, i.e., that are substantially free of all other diastereomers,without having to perform chiral isolation or separation steps. Becausethe reaction can be performed in high yield, utilizing highlyenantiopure 5-hydroxy-3-oxohexanoate ester (Ia) starting materials, thepresent disclosure enables the preparation of syn 3,5-dihydroxyhexanoateesters of structural formula (IIa) that are substantiallydiastereomerically pure with minimal or no purification. In contrast tothe present disclosure, prior reduction methods (e.g., U.S. Pat. No.6,596,879) produced 3-4% (IIb) (X═CN, R¹=t-butyl), which is an oil, andwhich was then reacted to form ((t-butyl6-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate) (X═CN, R¹=t-butyl,R³,R⁴=methyl), a crystalline compound. In the prior known methods, thepurity was improved by crystallization to lower the (IIb) content, whichresulted in yield losses of (IIa).

The high degree of diasteromeric purity of the reduction productsobtained with the engineered ketoreductase enzymes described herein isillustrated in FIG. 6, which compares the LC/MS/MS chromatogram oftert-butyl 6-cyanoethyl-(3R,5R)-dihydroxyhexanoate (structural formula(IIa), X═CN, R¹=t-butyl) prepared from commercially availablecrystalline t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-(4R)-acetate (structuralformula (IVa), X═CN, R¹=t-butyl, R³,R⁴=methyl) (Aldrich, St. Louis, Mo.)with that of crude tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate oilprepared from t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (structuralformula (Ia), X═CN, R¹=t-butyl) using an engineered ketoreductase enzymeof the present disclosure. As illustrated in FIG. 6, compared to atert-butyl (6R)-cyanoethyl-3,5-dihydroxyhexanoate sample prepared fromcommercially available crystalline t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-(4R)-acetate containing lessthan about 0.1% of the undesired (3S,5R) diastereomer (IIb), the crudetert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate oil prepared using theengineered ketoreductase of the disclosure contains no more thanpossibly 0.005% of the undesired (3S,5R) diastereomer.

Similarly striking differences in diastereomeric purity are observed fort-butyl (6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-(4R)-acetate (seeExample 23 and FIG. 7). GC/MS chromatograms comparing the stereoisomericpurity of the commercially available crystalline TBIN ((4R,cis)-1,1-dimethyl-6-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate)) andcrude, extracted, but never crystallized TBIN prepared from crude C7diol prepared using an engineered ketoreductase enzyme of the presentinvention are illustrated in FIG. 7. Likewise, the diastereomeric purityof atorvastatin produced from C7 diol prepared using the methods of thepresent disclosure are greater than what is currently present incommercial preparations (e.g., 0.03% atorvastatin diastereomer presentin a Lipitor® pill).

As used herein, a compound is “enriched” in a particular stereoisomerwhen that stereoisomer is present in excess over any other stereoisomerpresent in the compound. A compound that is enriched in a particularstereoisomer will typically comprise at least about 60%, 70%, 80%, 90%,or even more, of the specified stereoisomer. The amount of enrichment ofa particular stereoisomer can be confirmed using conventional analyticalmethods routinely used by those of skill in the art, as will bediscussed in more detail, below.

In some embodiments, the amount of undesired stereoisomers may be lessthan 10%, for example, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1% or even less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.2%, or0.1%. Stereoisomerically enriched compounds that contain at least about99.5% or more of the desired stereoisomer are referred to herein as“substantially pure” stereoisomers. In some embodiments, compounds thatare substantially pure in a specified stereoisomer contain greater than99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%,99.96%, 99.97%, 99.98% or even 99.99% of the particular stereoisomer.Stereoisomerically enriched compounds that contain ≧99.99% of thedesired stereoisomer are referred to herein as “pure” stereoisomers.

The stereoisomeric purity of any chiral compound described herein can bedetermined or confirmed using conventional analytical methods known inthe art. Highly sensitive LC/MS and GC/MS methods and for assessingdiastereoisomeric purity of the synthetic intermediates described hereinare provided in Examples 5-7. A method for determining diastereomericpurity of atorvastatin is given in Ertürk et al., 2003, J Pharm BiomedAnal. 33(5):1017-23.

As is known by those of skill in the art, ketoreductase-catalyzedreduction reactions typically require a cofactor. Reduction reactionscatalyzed by the engineered ketoreductase enzymes described herein alsotypically require a cofactor, although many embodiments of theengineered ketoreductases require far less cofactor than reactionscatalyzed with wild-type ketoreductase enzymes. As used herein, the term“cofactor” refers to a non-protein compound that operates in combinationwith a ketoreductase enzyme. Cofactors suitable for use with theengineered ketoreductase enzymes described herein include, but are notlimited to, NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Equation (1), below, illustrates anembodiment of a ketoreductase (“KRED”)-catalyzed reduction reactionutilizing NADH or NADPH as a cofactor, which are represented asalternatives by the designation NAD(P)H:

As illustrated, the reduced form of the cofactor is added to thereaction mixture. The reduced NAD(P)H form can be optionally regeneratedfrom the oxidized NAD(P)⁺ form using a cofactor regeneration system.

The term “cofactor regeneration system” refers to a set of reactantsthat participate in a reaction that reduces the oxidized form of thecofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the 5-hydroxy-3-oxo-hexanoate esterare regenerated in reduced form by the cofactor regeneration system.Cofactor regeneration systems comprise a stoichiometric reductant thatis a source of reducing hydrogen equivalents and is capable of reducingthe oxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD⁺ or NADP⁺, respectively, are known in the art and may be usedin the methods described herein.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acid and NADH orNADPH, respectively. Equation (2), below, describes the glucosedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by glucose.

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring, may be readily identified usingthe assay described in Example 4 of PCT publication WO 2005/018579, thedisclosure of which is incorporated herein by reference. Exemplarynon-naturally occurring glucose dehydrogenases are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 62, 64, 66, 68, 122, 124, and126. The polynucleotide sequences that encode them are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 61, 63, 65, 67, 121, 123, and125, respectively. All of these sequences are incorporated herein byreference. Additional non-naturally occurring glucose dehydrogenasesthat are suitable for use in the ketoreductase-catalyzed reductionreactions disclosed herein are provided in U.S. application publicationNos. 2005/0095619 and 2005/0153417, the disclosures of which areincorporated herein by reference.

Glucose dehydrogenases employed in the ketoreductase-catalyzed reductionreactions described herein may exhibit an activity of at least about 10μmol/min/mg and sometimes at least about 10² μmol/min/mg or about 10³μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in the assaydescribed in Example 4 of PCT publication WO 2005/018579.

When glucose and glucose dehydrogenase are employed as the cofactorregeneration system, as the 5-hydroxy-3-oxohexanoate ester substrate isreduced by the engineered ketoreductase and NADH or NADPH, the resultingNAD⁺ or NADP⁺ is reduced by the coupled oxidation of glucose to gluconicacid by the glucose dehydrogenase. The net reaction is described byequation (3), which is the summation of equations (1) and (2):

The ketoreductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not completelyinactivate the ketoreductase enzyme. Appropriate co-solvent systems canbe readily identified by measuring the enzymatic activity of thespecified engineered ketoreductase enzyme with the5-hydroxy-3-oxohexanoate ester of interest in the candidate solventsystem, utilizing an enzyme activity assay, such as those described inthe Examples.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. The reduction of the 5-hydroxy-3-oxohexanoateester to the corresponding 3,5-dihydroxyhexanoate ester can be carriedout at a pH of about 5 or above. Generally, the reduction is carried outat a pH of about 10 or below, usually in the range of from about 5 toabout 10. In some embodiments, the reduction is carried out at a pH ofabout 9 or below, usually in the range of from about 5 to about 9. Insome embodiments, the reduction is carried out at a pH of about 8 orbelow, often in the range of from about 5 to about 8, and usually in therange of from about 6 to about 8. The reduction may also be carried outat a pH of about 7.8 or below, or 7.5 or below. Alternatively, thereduction may be carried out a neutral pH, i.e., about 7.

During the course of the reduction reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range by the addition of an acid ora base during the course of the reaction. Alternatively, the pH may becontrolled by using an aqueous solvent that comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,for example, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used.

When the glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (3) causes the pH of the reaction mixture to drop if theresulting aqueous gluconic acid is not otherwise neutralized. The pH ofthe reaction mixture may be maintained at the desired level by standardbuffering techniques, wherein the buffer neutralizes the gluconic acidup to the buffering capacity provided, or by the addition of a baseconcurrent with the course of the conversion. Combinations of bufferingand base addition may also be used. Suitable buffers to maintain desiredpH ranges are described above. Suitable bases for neutralization ofgluconic acid are organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., K₂CO₃), bicarbonate salts (e.g., NaHCO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like. The addition of abase concurrent with the course of the conversion may be done manuallywhile monitoring the reaction mixture pH or, more conveniently, by usingan automatic titrator as a pH stat. A combination of partial bufferingcapacity and base addition can also be used for process control.

In such reduction reactions when the pH is maintained by buffering or byaddition of a base over the course of the conversion, an aqueousgluconate salt rather than aqueous gluconic acid is the product of theoverall process. For example, equation (4) represents the overallprocess when aqueous sodium hydroxide (Na⁺+OH⁻) is added over the courseof the reaction to maintain an initial pH below about 5 or above.

When base addition is employed to neutralize the gluconic acid releasedduring the ketoreductase-catalyzed reduction reaction, the progress ofthe conversion may be monitored by the amount of base added to maintainthe pH. Typically, bases added to unbuffered or partially bufferedreaction mixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

The terms “formate dehydrogenase” and “FDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of formate and NAD⁺ or NADP⁺ to carbon dioxide and NADH orNADPH, respectively. Formate dehydrogenases that are suitable for use ascofactor regenerating systems in the ketoreductase-catalyzed reductionreactions described herein include both naturally occurring formatedehydrogenases, as well as non-naturally occurring formatedehydrogenases. Formate dehydrogenases include those corresponding toSEQ ID NOS: 70 (Pseudomonas sp.) and 72 (Candida boidinii) of PCTpublication WO 2005/018579, which are encoded by polynucleotidesequences corresponding to SEQ ID NOS: 69 and 71, respectively, of PCTpublication 2005/018579, the disclosures of which are incorporatedherein by reference. Formate dehydrogenases employed in the methodsdescribed herein, whether naturally occurring or non-naturallyoccurring, may exhibit an activity of at least about 1 μmol/min/mg,sometimes at least about 10 μmol/min/mg, or at least about 10²μmol/min/mg, up to about 10³ μmol/min/mg or higher, and can be readilyscreened for activity in the assay described in Example 4 of PCTpublication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂ ⁻),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa (pKa=3.7in water) formate is present as both HCO₂ ⁻ and HCO₂H in equilibriumconcentrations. At pH values above about pH 4, formate is predominantlypresent as HCO₂ ⁻. When formate is provided as formic acid, the reactionmixture is typically buffered or made less acidic by adding a base toprovide the desired pH, typically of about pH 5 or above. Suitable basesfor neutralization of formic acid include, but are not limited to,organic bases, for example amines, alkoxides and the like, and inorganicbases, for example, hydroxide salts (e.g., NaOH), carbonate salts (e.g.,K₂CO₃), bicarbonate salts (e.g., NaHCO₃), basic phosphate salts (e.g.,K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂ ⁻, equation (5), below, describes the formatedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by formate.

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the 5-hydroxy-3-oxohexanoate ester is reduced bythe ketoreductase and NADH or NADPH, the resulting NAD⁺ or NADP⁺ isreduced by the coupled oxidation of formate to carbon dioxide by theformate dehydrogenase. An embodiment of the net reaction is described byequation (6), which is the summation of equations (1) and (5):

Equation (6) shows that when the formate/formate dehydrogenase cofactorregeneration system is employed for the reduction of5-hydroxy-3-oxohexanoate ester in aqueous solution at a pH above aboutpH 5, protons in solution are consumed and the reaction causes the pH ofthe reaction mixture to rise if it is not otherwise buffered orre-acidified. The pH of the reaction mixture may be maintained at thedesired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained. For example, equation (7) represents theoverall process when formic acid (HCO₂H) is added over the course of thereaction to maintain an initial pH above about pH 5. While the formateis present predominantly as HCO₂ ⁻ in the reaction mixture, the HCO₂ ⁻concentration is maintained while the conversion in net consumes theadded formic acid.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions.

The terms “secondary alcohol dehydrogenase” and “sADH” are usedinterchangeably herein to refer to an NAD⁺ or NADP⁺-dependent enzymethat catalyzes the conversion of a secondary alcohol and NAD⁺ or NADP⁺to a ketone and NADH or NADPH, respectively. Equation (8), below,describes the reduction of NAD⁺ or NADP⁺ by a secondary alcohol,illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the ketoreductase-catalyzed reduction reactionsdescribed herein include both naturally occurring secondary alcoholdehydrogenases, as well as non-naturally occurring secondary alcoholdehydrogenases. Naturally occurring secondary alcohol dehydrogenasesinclude known alcohol dehydrogenases from, Thermoanaerobium brockii,Rhodococcus erythropolis, Lactobacillus kefiri, and Lactobacillusbrevis, and non-naturally occurring secondary alcohol dehydrogenasesinclude engineered alcohol dehydrogenases derived therefrom. Secondaryalcohol dehydrogenases employed in the methods described herein, whethernaturally occurring or non-naturally occurring, may exhibit an activityof at least about 1 μmol/min/mg, sometimes at least about 10μmol/min/mg, or at least about 10² μmol/min/mg, up to about 10³μmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment the secondaryalcohol is isopropanol. Suitable aryl-alkyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, as the5-hydroxy-3-oxohexanoate ester substrate is reduced by the engineeredketoreductase and NADH or NADPH, the resulting NAD⁺ or NADP⁺ is reducedby the coupled oxidation of the secondary alcohol to the ketone by thesecondary alcohol dehydrogenase. The net reaction is described byequation (9), illustrated with isopropanol, which is the summation ofequations (1) and (8):

It will be appreciated by the skilled artisan this reaction of a ketoneand alcohol to the corresponding alcohol and ketone, respectively, isreversible, and will reach an equilibrium. The inventors found that theforward reaction is thermodynamically favored, and that only a modestexcess of isopropanol is required to obtain high conversion of the3-oxohexanoate ester to the desired 3,5-dihydroxyhexanoate ester at theequilibrium. Typically, the molar ratio of the secondary alcohol to the3-oxohexanoate ester provided to the reaction is in the range of 1 to20. In some embodiments this molar ratio is 1 to 10, 1 to 5, or 1 to 2.

Some engineered ketoreductases also have activity to dehydrogenate asecondary alcohol reductant. In some embodiments using secondary alcoholas reductant, the engineered ketoreductase and the secondary alcoholdehydrogenase are the same enzyme.

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the ketoreductase substrate (i.e., 5-hydroxy-3-oxohexanoateester) to the corresponding 3,5-dihydroxyhexanoate ester.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In carrying out the enantioselective reduction reactions describedherein, the engineered ketoreductase enzyme, and any enzymes comprisingthe optional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the engineered ketoreductase enzymeand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. For example,in some embodiments one set of host cells can be transformed withgene(s) encoding the engineered ketoreductase enzyme and another set canbe transformed with gene(s) encoding the cofactor regeneration enzymes.Both sets of transformed cells can be utilized together in the reactionmixture in the form of whole cells, or in the form of lysates orextracts derived therefrom. In other embodiments, a host cell can betransformed with gene(s) encoding both the engineered ketoreductaseenzyme and the cofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineeredketoreductase enzyme and/or the optional cofactor regeneration enzymes,or cell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of 3,5-dihydroxyhexanoateester desired, and concomitantly the amount of ketoreductase substrateemployed. The following guidelines can be used to determine the amountsof ketoreductase, cofactor, and optional cofactor regeneration system touse. Generally, 5-hydroxy-3-oxo-hexanoate ester substrates are employedat a concentration of about 20 to 300 grams/liter using from about 50 mgto about 5 g of ketoreductase and about 10 mg to about 150 mg ofcofactor. Those having ordinary skill in the art will readily understandhow to vary these quantities to tailor them to the desired level ofproductivity and scale of production. Appropriate quantities of optionalcofactor regeneration system may be readily determined by routineexperimentation based on the amount of cofactor and/or ketoreductaseutilized. In general, the reductant (e.g., glucose, formate,isopropanol) is utilized at levels above the equimolar level ofketoreductase substrate to achieve essentially complete or near completeconversion of the ketoreductase substrate.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, ketoreductase, and ketoreductase substrate may be added firstto the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, ketoreductase, and cofactor maybe added and mixed into the aqueous phase first. The organic phase maythen be added and mixed in, followed by addition of the ketoreductasesubstrate. Alternatively, the ketoreductase substrate may be premixed inthe organic phase, prior to addition to the aqueous phase

Suitable conditions for carrying out the ketoreductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredketoreductase enzyme and substrate at an experimental pH and temperatureand detecting product, for example, using the methods described in theExamples provided herein.

The ketoreductase catalyzed reduction is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or near complete, reduction of substrate is obtained.Reduction of substrate to product can be monitored using known methodsby detecting substrate and/or product. Suitable methods include gaschromatography, HPLC, and the like. Conversion yields of the3,5-dihydroxyhexanoate ester reduction product generated in the reactionmixture are generally greater than about 50%, may also be greater thanabout 60%, may also be greater than about 70%, may also be greater thanabout 80%, may also be greater than 90%, and are often greater thanabout 97%.

The 3,5-dihydroxyhexanoate reduction product can be recovered from thereaction mixture and optionally further purified using methods that areknown to those of skill in the art. Suitable methods are described inU.S. Pat. Nos. 6,001,615, 6,472,544, 6,596,879, and 6,645,746, thedisclosures of which are incorporated herein by reference. Additionalsuitable methods are provided in the Examples below.

As previously discussed, the ketoreductase-catalyzed reduction of the5-hydroxy-3-oxohexanoate ester enantiomer (Ia) generates a newstereogenic carbon at the 3-position of the 3,5-dihydroxyhexanoate esterproduct. As also previously discussed, in reactions carried out with theengineered ketoreductase enzymes described herein, the reduction productis generated with a high degree of syn-stereoselectivity at the3-position: Thus, the 3,5-dihydroxyhexanoate esters generated using theengineered ketoreductase enzymes described herein are chiral andnon-racemic. As mentioned above, the ketoreductase reactions describedherein typically generate chiral syn 3,5-dihydroxyhexanoate estersaccording to structural formula (IIa) having a diastereomeric purity ofat least about 99.5%, usually at least about 99.9%, and typically atleast about 99.95%. The Examples illustrate embodiments providingtert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ester with adiastereomeric purity of ≧99.993%.

Certain of the substrates described herein have been found to beparticularly useful in the synthesis of clinically importantcholesterol-lowing statin drugs. Such compounds include those in whichR¹ is a lower alkyl, in some embodiments, tert-butyl, and X is selectedfrom chloro, cyano and hydroxyl and OR² is as defined herein. Thus, someembodiments of the compounds according to structural formulae (Ia),(IIa), (IIIa), (IVa) and (Va) described herein include those compoundsin which R¹ is a lower alkyl or tert-butyl and/or X is selected fromchloro, cyano and hydroxy.

In embodiments of the compounds according to structural formulae (Ia),(IIa), (IIIa), (IVa) and (Va) in which X is —OR⁶, where R⁶ is aprotecting group, the group can be virtually any protecting group knownto be useful for protecting hydroxyl groups during organic syntheses.The identity of the specific protecting group will depend upon thevarious synthetic reaction conditions, and can be readily selected forspecific situation by those of skill in the art. Suitable exemplaryprotecting groups are described in Greene & Wuts, “Protective Groups inOrganic Synthesis,” 3^(rd) Ed., 1999, John Wiley & Sons, NY (see, forexample, pp. 17-200). In some embodiments, the protecting group isselected from ether protection groups (e.g., benzyl, t-butyl,tetrahydropyranyl, methoxyethoxymethyl, methoxymethyl, etc.), silylether protection groups (e.g., trimethylsilyl, triethylsilyl,t-butyl-dimethylsilyl, triphenylsilyl, etc.), ester protection groups(e.g., benzoate, alkylate, haloalkylate, etc.), carbonate protectinggroups (e.g., alkyl methyl, methoxymethyl, alkyl 2,2,2,-trichloroethyl,allyl allyl carbonate, alkyl benzyl, etc.), and sulfonate protectinggroups (e.g., allylsulfonate, methanesulfonate, benzylsulfonate,tosylate, etc.).

The 5-hydroxy-3-oxohexanoate ester enantiomers of structural formulae(Ia) can be prepared using standard methods that are well-known in theart. Suitable exemplary methods are described in U.S. Pat. No. 6,399,339(for specific examples in which X═Cl and R¹=tert-butyl, see Example 1);U.S. Pat. No. 5,155,251 and the various references cited therein (X═CN,R¹=tert-butyl), Japanese Patent Publication No. 1723728 (X═Cl,R¹=tert-butyl) and PCT publication WO 2005/018579 (X═Cl or CN,R¹=tert-butyl), the disclosures of which are incorporated herein byreference. FIG. 1 provides a diagram illustrating exemplary methods forsynthesizing the substrates in both enantiomerically pure form and asracemic mixtures (International Publication Nos. WO 2003/070733 and WO2004/113314).

The syn 3,5-dihydroxyhexanoate esters produced by the engineeredketoreductase-catalyzed reduction reactions described herein can be usedas starting materials to synthesize clinically importantcholesterol-lowering statin drugs, such as atorvastatin, rosuvastatin,and pitavastatin. A key intermediate in the synthesis of these statinsis the protected syn 3,5-dihydroxyhexanoate ester of structural formula(IIIa) or (IVa), supra. Methods for synthesizing such protected estersare well known. In embodiments in which the R² substituents in thecompounds of structural formula (IIIa) represent unbridged protectinggroups, the protecting groups can be selected from those describedabove. Compounds including these protecting groups can be synthesized asdescribed, for example, in Greene & Wuts, “Protective Groups in OrganicSynthesis,” 3^(rd) Ed., 1999, John Wiley & Sons, NY (see, for example,pp. 17-200). The actual methods used will depend upon the identity ofthe protecting group(s).

Embodiments in which the R² groups are taken together to form anoptionally substituted alkylene bridge can be synthesized from the syn3,5-dihydroxyhexanoate esters using the methods described in U.S. Pat.No. 5,097,045 (X=cyano), U.S. Pat. No. 6,472,544 (X=halo or OH) and U.S.Pat. No. 6,344,569 (X=halo or CN), the disclosures of which areincorporated herein by reference. Compounds including these protectinggroups can be synthesized as described, for example, in Greene & Wuts,“Protective Groups in Organic Synthesis,” 3rd Ed., 1999, John Wiley &Sons, NY (see, for example, pp. 201-245).

As a specific example, compounds according to structural formula (IVa)in which X is cyano can be prepared by treating the corresponding(3R,5R)-6-cyano-3,5-dihydroxyhexanoate ester with an acetal formingreagent as described in U.S. Pat. No. 6,344,569, at Col. 7, line 30through Col. 8, line 40, the disclosure of which is incorporated hereinby reference.

As another specific example, compounds according to structural formula(IVa) in which X is halo can be prepared by treating the corresponding(3R,5S)-6-halo-3,5-dihydroxyhexanoate ester with an acetal formingreagent as described in U.S. Pat. No. 6,344,569 at Col. 9, line 21through Col. 10, line 34, the disclosure of which is incorporated hereinby reference.

As yet another specific example, compounds according to structuralformula (IVa) in which X is cyano can be prepared by treating the chloroanalog (prepared as described above), with a cyanating reagent asdescribed in U.S. Pat. No. 6,344,569 at Col. 11, line 17 through Col.12, line 5, the disclosure of which is incorporated herein by reference.

In some embodiments, the R² groups of the protected syn3,5-dihydroxyhexanoate taken together can form a cyclic boronateprotecting group of —BR⁵—, wherein the R⁵ is selected from (C1-C12)alkyl, (C6-C10) aryl and (C7-C12) arylalkyl. In a specific embodiment,R⁵ is phenyl. Compounds with cyclic boronate based protecting groups canbe synthesized as described, for example, in Greene & Wuts, “ProtectiveGroups in Organic Synthesis,” 3rd Ed., 1999, John Wiley & Sons, NY (see,for example, pp. 243-245). An exemplary phenyl boronate protected syn3,5-dihydroxyhexanoate ester is described in U.S. Pat. No. 6,867,306,incorporated herein by reference.

Exemplary embodiments of these and other reactions are illustrated inFIG. 2. Specific methods of synthesizing tert-butyl(4R,6R)-6-cyanomethyl-2,2-dimethyl1-1,3-dioxane-4-acetate fromtert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate and tert-butyl(4R,65)-6-chloromethyl-2,2-dimethyl-1,3-dioxane-4-acetate fromtert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate are described inExample 2 and 3, respectively, of U.S. Pat. No. 6,344,569, thedisclosure of which is incorporated herein by reference. A specificmethod for synthesizing tert-butyl(4R,6R)-6-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate from tert-butyl(4R,6S)-6-chloromethyl-2,2-dimethyl-1,3-dioxane-4-acetate is describedin Example 3 of U.S. Pat. No. 6,344,569, the disclosure of which isincorporated herein by reference.

The protected and unprotected syn dihydroxy esters of structuralformulas (IIa), (IIIa), (IVa), and (Va) can be used to synthesizecholesterol lowering statins, as well as other useful compoundsincluding a 1,3-syn dihydroxy moiety, using methods that are well-knownin the art. For example, methods of synthesizing atorvastatin usingtert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate are described in U.S.Pat. No. 6,596,879 (see especially Example 2 at Col. 14, line 22-58),and U.S. Pat. No. 5,097,045, the disclosures of which is incorporatedherein by reference.

Methods of synthesizing rosuvastatin using tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate are described in U.S. Pat. No.5,278,313 and U.S. Pat. No. 6,472,544, respectively, the disclosures ofwhich are incorporated herein by reference. Methods of synthesizingrosuvastatin using tert-butyl (3R,5S,6)-trihydroxyhexanoate aredescribed in PCT application no. WO 01/85975, the disclosure of which isincorporated herein by reference. Synthesis of other statins using thecompounds produced by the methods herein will be apparent to the skilledartisan.

8. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

In the following description, wherever glucose dehydrogenase (GDH) isused, it is GDH-CDX901, obtainable from Julich Chiral Solutions, Jülich,Germany.

8.1 Example 1 Wild-Type Ketoreductase Gene Acquisition and Constructionof Expression Vectors

The Ydl124wp gene (Genbank Acc. No.: NP_(—)010159.1; GI: 6320079)encoding a Saccharomyces cerevisiae ketoreductase was codon optimized(SEQ ID NO:1) for expression in E. coli based on the amino acid sequence(SEQ ID NO:2) of the ketoreductase (“YDL”) encoded by the Ydl124wp gene.The gene was synthesized using oligonucleotides composed of 42nucleotides and cloned into expression vector pCK110900 (depicted inFIG. 5) under the control of a lac promoter. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Resulting plasmids were transformed into E. coli W3110using standard methods. Several clones were selected for sequencing theputative KRED-encoding gene. Genes having the desired designed sequencewere identified and used as the starting material for the development ofgenes encoding engineered ketoreductases.

This Example illustrates the acquisition of a recombinant polynucleotideencoding the wild-type ketoreductase YDL from which genes encodingengineered ketoreductases can be derived, and expression vectors andhost cells suitable for such engineering.

Polynucleotides encoding engineered ketoreductases of the presentdisclosure were likewise cloned into vector pCK110900 for expression inE. coli W3110.

8.2 Example 2 Production of Ketoreductase Powders; Shake Flask Procedure

A single microbial colony of E. coli containing a plasmid with theketoreductase gene of interest was inoculated into 50 ml Luria Bertanibroth containing 30 μg/ml chloramphenicol and 1% glucose. Cells weregrown overnight (at least 16 hrs) in an incubator at 30° C. with shakingat 250 rpm. The culture was diluted into 250 ml Terrific Broth (12 g/Lbacto-tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO₄, 30 μg/ml chloramphenicol) in 1 literflask) to an optical density at 600 nm (OD₆₀₀) of 0.2 and allowed togrow at 30° C. Expression of the ketoreductase gene was induced with 1mM IPTG when the OD₆₀₀ of the culture is 0.6 to 0.8 and incubatedovernight (at least 16 hrs). Cells were harvested by centrifugation(5000 rpm, 15 min, 4° C.) and the supernatant discarded. The cell pelletwas resuspended with an equal volume of cold (4° C.) 100 mMtriethanolamine (chloride) buffer, pH 7.0, and harvested bycentrifugation as above. The washed cells were resuspended in twovolumes of the cold triethanolamine (chloride) buffer and passed througha French Press twice at 12000 psi while maintained at 4° C. Cell debriswas removed by centrifugation (9000 rpm, 45 min., 4° C.). The clearlysate supernatant was collected and stored at −20° C. Lyophilization offrozen, clear lysate provided a dry powder of crude ketoreductaseenzyme.

This Example illustrates the production of ketoreductases powderssuitable for use to test enzyme properties.

8.3 Example 3 Preparation of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate

To an 1 L 3-neck flask with an addition funnel and a PTFE-coatedmagnetic stirring bar, under a nitrogen atmosphere and immersed in a−20° C. dry ice/acetone bath, was added 120 mL anhydrous tetrahydrofuranand 75 mL (540 mmol) diisopropylamine. The solution was stirred andcooled to −20° C., and 200 mL (500 mmol) 2.5 M n-butyllithium in hexanewas added dropwise over 45 minutes, giving a pale yellow solution. Afteran additional 30 minutes, 70 mL (520 mmol) t-butyl acetate was addeddropwise with stirring over 25 minutes, turning the solution cloudy.After an additional 1 hour maintained at −20° C., a solution of 22.0 gethyl 4-cyano-(3R)-hydroxybutyrate (>99.9% e.e., 140 mmol) in 30 mLanhydrous tetrahydrofuran was added dropwise with stirring over 35minutes, giving a pale yellow solution. The solution was maintained at−20° C. for an additional 1 hour, then transferred via cannula over 30minutes subsurface into a stirred biphasic mixture of 350 mL 1 M citricacid and 250 mL toluene immersed in an ice bath (˜5° C.). During thisquench, the temperature of the mixture was maintained between 5-10° C.Upon completion, the phases separated, giving a pale yellow organicphase above and an orange aqueous phase, pH ˜6, below. The aqueous phasewas separated and extracted with 250 mL toluene. The combined organicphases were washed with 100 mL 1 N HCl followed by 3×100 mL of distilledwater. The final water wash gave pH ˜5. The solvent was removed from theorganic phase by rotary evaporation at 60° C. under vacuum to give 31.7g of a yellow oil.

¹H-NMR of the oil showed the presence of t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate, t-butyl 3-oxobutyrate, and t-butyl4-cyano-(3R)-hydroxybutyrate in 1.00:0.27:0.14 mole ration,corresponding to 77 weight % t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoateif no other components are present. The actual weight % was likelyslightly lower, but was not more accurately measured. 77 weight % of31.7 g oil corresponds to a 77% mole yield on ethyl4-cyano-(3R-hydroxybutyrate (theoretical yield=31.8 g).

This and similarly prepared crude t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate, typically having purity rangingfrom 60 to 75 weight %, the balance being t-butyl 3-oxobutyrate andt-butyl 4-cyano-(3R)-hydroxybutyrate, were used in the followingExamples, except when the purified substrate is specified. The specifiedamounts of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate used in theExamples refer to the amount of the crude preparation, uncorrected forits actual composition.

8.4 Example 4 LC/MS/MS Assay for Substrate Specificity and Conversion

An LC/MS/MS method was developed to analyze the conversion of t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate and the possible conversion oft-butyl 3-oxobutyrate in the crude substrate (Example 4) to t-butyl3-hydroxybutyrate:

Instrument: Agilent 1100 with Waters Micromass Quattro Premier LC/MS/MS

Column: Agilent SB C18 (50 mm×4.6 mm, 5 nm)

Mobile phase: 50% aqueous 0.25 vol. % acetic acid, 50% acetonitrile

Flow rate: 1.0 mL/min.

Backpressure: 60 bar

Temperature: ambient room temperature

Injection: 5 μL, ˜0.1 mg/ml in methanol or other water miscible solvent.

Retention t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate 1.0 min. times:t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate 1.3 min. t-butyl3-hydroxybutyrate 1.5 min. t-butyl 3-oxobutyrate 2.1 min.

Ionization/Detection: APCI+ using the transitions MH+>MH-56 (loss oft-butene)

The response ratio for t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate tot-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate substrate was typically 0.84at 0.1 mg/ml. The MS/MS transitions for t-butyl 3-oxobutyrate andt-butyl 3-hydroxybutyrate were not optimized.

8.5 Example 5 Diastereomeric Analysis oft-butyl-6-cyano-3,(5R)-dihydroxyhexanoate by LC/MS/MS

An LC/MS/MS method was developed to separate and analyze the (3R,5R) and(3S,5R) diastereomers of t-butyl 6-cyano-3,(5R)-dihydroxyhexanoate. TheLC conditions provided baseline resolution of the diastereomers. Theresponse was linear from 0.0001 to 1.0 mg/ml with a detection limitunder 0.0001 mg/ml.

Instrument: Agilent 1100 with Waters Micromass Quattro Premier LC/MS/MS

Column: Agilent SB C8 (50 mm×4.6 mm, 5 μm)

Mobile phase: Isocratic 65% of 0.25% aq acetic acid, 35% acetonitrile

Flow rate: 1.0 mL/min.

Temperature: ambient room temperature

Retention times: (3S,5R) 12.77 min: (3R,5R) 13.67 min.

Ionization/Detection: APCI+ using the transitions MH+>MH-56 (loss oft-butene)

8.6 Example 6 Diastereomeric Analysis of t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate by GasChromatography

A GC/MS method was developed to separate and analyze the (4R,6R) and(4S,6R) diastereomers of t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate:

Column: HP5MS (0.25 mm i.d., 25 μm df, 30 m)

Carrier gas: Helium, 9.44 psi, 0.7 mL/minute.

Oven: 60° C. for 1 min, then 15° C./min to 280° C., then hold at 280° C.for 2 min.

Retention Times: (4S,6R) 11.44 min; (4R,6R) 11.55 min.

Detection: total ion count

8.7 Example 7 Diastereomeric Analysis oft-butyl-6-cyano-3,(5R)-dihydroxyhexanoate via derivatization to t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate

To a 30 mg sample comprising t-butyl 6-cyano-3,(5R)-dihydroxyhexanoatewas added 1 mL of ethyl acetate, 200 μL of 2,2-dimethoxypropane, and 20μL of methanesulfonic acid. The mixture was stirred for 20 minutes atroom temperature. After addition of 200 μL of saturated aqueous sodiumbicarbonate, a sample of the top ethyl acetate layer was analyzed by themethod of Example 6.

Each of the (3R,5R) and (3S,5R) diastereomers oft-butyl-6-cyano-3,(5R)-dihydroxyhexanoate are quantitatively derivatizedunder these conditions. Accordingly the 3,(5R) diastereomericcomposition of the t-butyl-6-cyano-3,(5R)-dihydroxyhexanoate correspondsto the analyzed 4,(6R) diastereomeric composition of the(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-4-acetate derivative.

8.8 Example 8 Evaluation of Wild-Type Ketoreductase YDL for Activity toReduce t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate

To a reaction mixture of 950 μL 100 mM triethanolamine (chloride)buffer, pH 7.0, 200 μL isopropanol, 9 mg t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate and 8 mg NAD(P)H was added 15 mgketoreductase powder comprising ketoreductase YDL. The reaction mixturewas stirred at room temperature for 24 hours, then extracted with 1 mLethyl acetate. After centrifugation, the organic phase was separated andthe solvent was removed by rotary evaporation to leave a nonvolatileoil. The nonvolatile oil was analyzed by the method of Example 4 for theconversion of the t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate and, ifpresent, the diastereomeric composition of the product, t-butyl6-cyano-(3,5R)-dihydroxyhexanoate by the method of Example 7. Theanalyses showed 85% conversion of the t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to produce t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate having >99% d.e.

The Example illustrates the identification of wild-type ketoreductaseYDL as a candidate for engineering to improve properties for thereduction of a 3-oxo group of an enantiomer of a5-hydroxy-3-oxohexanoate ester to yield the corresponding syn3,5-dihydroxyhexanoate ester.

8.9 Example 9 Evaluation of Wild-Type Ketoreductase YDL for Activity toReduce t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate Using Glucose andGlucose Dehydrogenase for Cofactor Regeneration

To a reaction mixture of 1 mL 100 mM triethanolamine (chloride) buffer,pH 7.0, 100 μL butyl acetate, 20 mg t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate, 1 mg NAD(P)⁺, and 35 mg glucose,was added 3 mg of glucose dehydrogenase powder and 20 mg ketoreductasepowder comprising ketoreductase YDL. The reaction mixture was stirred atroom temperature for 22 hours, then extracted with 1 mL ethyl acetate.After centrifugation, the organic phase was separated and the solventwas removed by rotary evaporation to leave a nonvolatile oil. Thenonvolatile oil was analyzed by the method of Example 4 for theconversion of the t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate and, ifpresent, the diastereomeric composition of the product, t-butyl6-cyano-(3,5R)-dihydroxyhexanoate by the method of Example 5. Theanalyses showed 92.6% conversion of the t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to produce t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate having >99.9% d.e.

The Example illustrates the evaluation of wild-type ketoreductase YDLfor activity and diastereoselectivity when used in combination with acofactor regeneration system (glucose and glucose dehydrogenase).

8.10 Example 10 Evaluation of Substrate and Cofactor Specificities ofWild-Type Ketoreductase YDL in the Reaction to Reduce Crude t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate

The specificity of wild-type ketoreductase YDL powder for the reductionsof t-butyl-(R)-5-hydroxy-3-oxohexanoate and of t-butyl 3-oxobutyrate byNADH and NADPH was evaluated by measuring the increase in NAD(P)absorbance at 340 nm in reaction mixtures comprising 3 mg/mL purifiedt-butyl-(R)-5-hydroxy-3-oxohexanoate or ˜13 mM t-butyl 3-oxobutyrate and0.2-0.3 mM NADH or NADPH in the total volume of 1 mL 100 mMtriethanolamine (chloride) buffer, pH 7.2.

Ketoreductase YDL powder was active for the reduction oft-butyl-(R)-5-hydroxy-3-oxohexanoate by NADPH but not by NADH.Ketoreductase YDL powder showed only very low activity for the reductionof t-butyl 3-oxobutyrate by NADPH, relative to that ont-butyl-(R)-5-hydroxy-3-oxohexanoate.

8.11 Example 11 High Throughput Assay for Ketoreductase Activity ont-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate

A. High Throughput NAD(P)H Fluorescence Assay: Library colonies werepicked using a Q-bot® robotic colony picker (Genetix USA, Inc.,Beaverton, Oreg.) into 96-well shallow well microtiter plates containing180 μL Luria Bertani broth (LB), 1% glucose and 30 μg/mL chloramphenicol(CAM). Cells were grown overnight at 30° C. with shaking at 250 rpm. 13μL of this culture was then transferred into 96-deep well platescontaining 400 μL Terrific broth (TB) and 30 μg/mL CAM. After incubationof deep-well plates at 30° C. with shaking at 250 rpm for 2.5 to 3 hours(OD₆₀₀ 0.6-0.8), recombinant gene expression by the cell cultures wasinduced by isopropyl thiogalactoside (IPTG) to a final concentration of1 mM. The plates were then incubated at 30° C. with shaking at 250 rpmfor overnight.

Cells were pelleted via centrifugation, resuspended in 300 to 400 μLlysis buffer and lysed by shaking at room temperature for at least 2hours. The lysis buffer contained 100 mM triethanolamine (chloride)buffer, pH 7.0-7.2, 1 mg/mL lysozyme and 500 μg/mL polymixin B sulfate.

Ketoreductase activity was measured by transferring measured quantitiesof the lysis mixtures into the wells of microtiter plates containing anassay mixture of 100 mM triethanolamine (chloride) buffer, pH 7-7.2,0.2-0.3 mM NAD(P)H, 2 to 3 mg/mL t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate, 0 to 2 mg/ml t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate, 0 to 600 mM glucose and 0 to 600 mMsodium gluconate. The reactions were monitored by measuring the decreaseof fluorescent emission of NAD(P)H at 440 nm as a function of time. Theresults were plotted as relative fluorescent units (RFU) of NAD(P)Hverses time and the slope of the plot (RFU/min) was used to determinethe rate of reaction.

B. Medium Throughput LC/MS/MS Assay for Substrate Specificity andConversion: To determine the extent to which the NAD(P)H consumptionmeasured in the fluorescence assay was due to conversion of the t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate substrate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate product or to reduction of t-butyl3-oxobutyrate in the crude substrate to t-butyl 3-hydroxybutyrate, thereaction mixtures showing NAD(P)H consumption activity of interest wereconsolidated into fewer 96-well microtiter plates and extracted withethyl acetate. The separated ethyl acetate extracts were diluted withacetonitrile/water (1/1) and analyzed by the LC/MS/MS method of Example4.

8.12 Example 12 High Throughput Screen for More Stable EngineeredKetoreductases

Lysis mixtures comprising engineered ketoreductases prepared andidentified as in Example 11A were incubated for at least 16 hrs at atemperature in the range of 37° C. to 50° C. The ketoreductase activityremaining in the lysis mixtures after the incubation was then measuredat ambient temperature by the procedure of Example 4. The ratio of theketoreductase activity remaining after the incubation to the activity ofthe corresponding unincubated lysis mixture, 0 to 1, was used a measureof the stability of the engineered ketoreductase in the lysis mixture.

8.13 Example 13 Improved Activity of Engineered Ketoreductases Derivedfrom Wild-Type YDL for the Reduction of t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate

To a 25 mL three-neck vessel equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into thevessel, was charged 19 mL 100 mM triethanolamine (chloride) buffer, pH7, 3.0 g glucose, ketoreductase powder comprising the ketoreductasespecified by SEQ ID NOS. in Table 2 in the amount specified in Table 2,13 mg glucose dehydrogenase powder, 1.0 mg NaNADP, and lastly 3 gt-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate. The reaction mixture wasstirred at 22° C. The automatic titrator maintained the pH at 7 by theaddition of 4N NaOH, which was continuously recorded. Reaction progresswas monitored by the rate and cumulative addition of the base andperiodic sampling of the reaction mixture for extraction and analysis bythe method of Example 4. After the reaction time specified in Table 2,the reaction mixture was extracted twice with 10 mL ethyl acetate twice.The combined organic extract was filtered through a short pad ofdiatomaceous earth and the solvent was removed by rotary evaporationunder vacuum. Diastereomeric analysis of the residual oil by the methodof Example 5 showed the product t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate from each such reaction reportedTable 2 to have >99.99% diastereopurity. (No other diastereomers weredetectable.)

Table 2 gives the SEQ ID NOS. corresponding to the ketoreductase powder,the number of amino acid mutations from the wild-type ketoreductase YDL,the amount of ketoreductase powder used, the reaction time, and theconversion of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate reached in that reaction time.

This Example illustrates, by comparisons of ketoreductase amounts,reaction times, and conversions in Table 2, engineered ketoreductasesderived from the wild-type ketoreductase YDL providing improved activitycompared to ketoreductase YDL.

TABLE 2 Activities of engineered ketoreductases derived from YDL.ketoreductase mutations ketoreductase reaction conversion SEQ ID NO.from YDL amount (mg) time (h) (%) 2 (YDL) 0 120 67 87 70 3 120 67 99.6112 4 60 24 94 178 7 30 24 95 276 8 30 23 96 278 8 30 22 99.0 318 9 30 599.7

8.14 Example 14 Improved Stability of Engineered Ketoreductases Derivedfrom Wild-Type YDL for the Reduction of t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate

To a 25 mL three-neck vessel equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into thevessel, was charged 20 mL 100 mM triethanolamine (chloride) buffer, pH7, 0.5 g glucose, the amount of ketoreductase powder specified in Table3, 8 mg glucose dehydrogenase, 2.0 mg NaNADP, and lastly 0.3 g t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate. The reaction mixture was stirred atthe temperature specified in Table 3. The automatic titrator maintainedthe pH at 7 by the addition of 4N NaOH, which was continuously recorded.Reaction progress was monitored by the rate and cumulative addition ofthe base and periodic sampling of the reaction mixture for extractionand analysis by the method of Example 4. After 22 hours, the reactionmixture was extracted twice with 10 mL ethyl acetate twice. The combinedorganic extract was filtered through a short pad of diatomaceous earthand the solvent was removed by rotary evaporation under vacuum. Analysisof the residual oil by the method of Example 5 showed the productt-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate from each such reactionreported Table 3 having >99.99% diastereopurity. (No other diastereomerswas detectable.)

Table 3 gives the SEQ ID NOS. corresponding to the ketoreductase powder,the number of amino acid mutations from the wild-type ketoreductase YDL,the amount of ketoreductase powder used, the temperature of thereaction, and the conversion of t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate to t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate reached at the specified cumulativereaction time.

TABLE 3 Activities of YDL and engineered ketoreductases derived fromYDL. ketoreductase mutations ketoreductase temperature conversion (%)SEQ ID NO. from YDL amount (mg) (° C.) at 3 hours at 17 hours at 22hours 2(YDL) 0 30 22 29 50 75 2(YDL) 0 30 30 25 25 25 154 7 30 30 43 7878 312 11 30 30 40 90 >99 316 9 10 30 33 92 96

This Example illustrates, by comparisons of the ketoreductase amounts,reaction times, and conversions in Table 3 engineered ketoreductasesderived from the wild-type ketoreductase YDL providing improvedstability compared to YDL. While in the reaction at 22° C., YDL retainedactivity and the reaction reached 75% conversion in 22 hours, in thereaction at 30° C., YDL lost all activity within 3 hours reaching only25% conversion. The engineered ketoreductase having the amino acidsequence SEQ ID NO. 156 is partially improved in stability; it retainedbeyond 3 hours at 30° C., but lost all activity within 17 hours, havingreached 78% conversion. The engineered ketoreductase having the aminoacid sequence SEQ ID NO. 312 retained activity beyond 17 hours at 30° C.and reached essentially complete conversion within 22 hours.

8.15 Example 15 Improved Activity and Diastereoselectivity of EngineeredKetoreductases Derived from Wild-Type YDL for the Reduction of t-butyl6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl6-chloro-(3R,5S)-dihydroxyhexanoate

To a 50 mL three-neck vessel equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into thevessel, was charged 21 mL 100 mM triethanolamine (chloride) buffer, pH7, 0.5 g glucose, 2 mL of the same buffer containing 10 mg ofketoreductase powder comprising the ketoreductase specified by SEQ IDNO. in Table 4, 10 mg glucose dehydrogenase powder, 2.0 mg NAD orNaNADP, and lastly 0.3 g t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate.The reaction mixture was stirred at 22° C. The automatic titratormaintained the pH at 6.9 by the addition of 4N NaOH, which wascontinuously recorded. Reaction progress was monitored by the rate andcumulative addition of the base and periodic sampling of the reactionmixture for extraction and analysis by the method of Example 4. Afterthe reaction time specified in Table 4, the reaction mixture wasextracted twice with 10 mL ethyl acetate twice. The combined organicextract was filtered through diatomaceous earth and the solvent wasremoved by rotary evaporation under vacuum to give 0.2 g residual oil.

Diastereopurity of the t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoateproduct in the residual oil was determined by the method of Example 7.The (3R,5S)-diol is a syn diastereomer. (The convention for labeling the5-position R or S is reversed between 6-cyanomethyl and 6-chloromethyl.)

Table 4 gives the SEQ ID NOS. corresponding to the ketoreductase powder,the number of amino acid mutations from the wild-type ketoreductase YDL,the amount of ketoreductase powder used, the cofactor used, the reactiontime, the conversion of t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate,and the diastereopurity of the t-butyl6-chloro-(3R,5S)-dihydroxyhexanoate product.

This Example illustrates an engineered ketoreductases derived from thewild-type ketoreductase YDL providing improved activity and improveddiastereoselectivity compared to ketoreductase YDL and the use of suchengineered ketoreductases for the preparation of t-butyl6-chloro-(3R,5S)-dihydroxyhexanoate.

TABLE 4 Activities and stereoselectivities of YDL and engineeredketoreductases derived from YDL to produce t-butyl6-chloro-(3R,5S)-dihydroxyhexanoate. ketoreductase mutations reactionconversion d.e.(%) cofactor SEQ ID NO. from YDL time (h) (%) (3R,5S)NADP 2(YDL) 0 20 37 97.3 326 10 4 99.8 99.2 328 11 4 99.6 99.4 NAD2(YDL) 0 20 28 98.9 326 10 5 99.5 99.8 328 11 20 98.5 99.6

8.16 Example 16 Reduction of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoatet-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by an EngineeredKetoreductase Using Isopropanol and a Secondary Alcohol Dehydrogenasefor Cofactor Regeneration

To a reaction mixture of 2 mL 100 mM triethanolamine (chloride) buffer,pH 7.2, containing 2 mM MgSO₄, 360 mg t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate, and 200 μL isopropanol, were added30 mg ketoreductase powder comprising the engineered ketoreductasehaving the amino acid sequence of SEQ ID NO. 330, secondary alcoholdehydrogenase powder comprising an engineered secondary alcoholdehydrogenase derived from ADH-LK and having activity to oxidizeisopropanol to acetone, and 5 mg NaNADP. The reaction mixture wasstirred at room temperature for 1 hour, then extracted with 1 mL ethylacetate. After centrifugation, the organic phase was separated and thesolvent was removed by rotary evaporation to leave a nonvolatile oil.The nonvolatile oil was analyzed by the methods of Examples 4 and 7. Thet-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate was 100% converted tot-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate with >99.9% d.e. (=>99.95%diastereoselectivity).

This Example illustrates the reduction of the 3-oxo group of anenantiomer of a 5-hydroxy-3-oxohexanoate ester to yield thecorresponding syn 3,5-dihydroxyhexanoate ester using an engineeredketoreductase in combination with a cofactor regeneration systemcomprising a secondary alcohol (isopropanol) and a secondary alcoholdehydrogenase.

8.17 Example 17 Preparation of t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate (a Compound of Structural FormulaIIa)

To a 100 mL three-neck flask equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into theflask, was charged 19 mL 100 mM triethanolamine (chloride) buffer, pH 7,8.68 g glucose, 100 mg ketoreductase powder comprising the engineeredketoreductase having the amino acid sequence SEQ ID NO. 316, 40 mgglucose dehydrogenase powder, 6 mg NaNADP, and lastly 12 g t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate. The reaction mixture was stirred at22° C. The automatic titrator maintained the pH at 6.9 by the additionof 4N NaOH, which was continuously recorded. After 22 hours, 8.9 ml 4 NNaOH had been consumed, and an extract of a sample analyzed by themethod of Example 4 showed >99% conversion. The reaction mixture wasfiltered through diatomaceous earth and 20 mL ethyl acetate was addedthrough the diatomaceous earth. The aqueous phase was separated andextracted with 20 mL ethyl acetate. The solvent was removed from thecombined organic extracts by rotary evaporation under vacuum to obtain9.80 g crude t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate as an oil.

This Example illustrates the preparation and recovery of a syn3,5-dihydroxyhexanoate ester using an engineered ketoreductase by themethod of the invention.

8.18 Example 18 Preparation of t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-(4R)-acetate (a Compound ofStructural Formula IVa)

To the 9.80 g crude t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate obtainedin Example 17 was added 26 mL 2,2-dimethoxypropane and 260 μLmethanesulfonic acid. The mixture was stirred at for 2 hours at 22° C.After addition of 25 mL 5% (half saturated) aqueous sodium bicarbonate,the pH of the aqueous phase was 7.5. The mixture was extracted with 52mL heptane, and the separated organic phase was extracted with 25 mL 5%aqueous sodium bicarbonate. The solvents were removed from the organicphase by rotary evaporation under vacuum. The residue was taken up in 70mL heptane with mild heating (˜45° C.), cooled and maintained at roomtemperature for 30 minutes, then cooled maintained at 0° C. to completethe crystallization. Filtration and drying yielded at 8.1 g t-butyl(6R)-cyanomethyl-2,2-dimethyl-1,3-dioxane-(4R)-acetate as a slightlyyellow solid. Analysis by the method of Example 6 showed 97.8 area %purity and no detectable (4S,6R) diastereomer.

This Example illustrates the preparation and recovery of a protected syndihydroxy hexanoate ester from a syn 3,5-dihydroxyhexanoate esterprepared by the method of the invention.

8.19 Example 19 Preparation of t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate

To a jacketed 3 L three-neck flask equipped with a PTFE-coated magneticstirring bar, an addition funnel, and a pH electrode connected to anautomatic titrator for pH-controlled addition of base on-demand via afeeding tube into the flask, was charged 6.36 g triethanolamine and 427mL water. The pH of the solution was adjusted to 6.9 by addition of 3.85g concentrated hydrochloric acid. 195 g D-glucose, 0.90 g glucosedehydrogenase powder, 2.25 g ketoreductase powder comprising theengineered ketoreductase having the amino acid sequence SEQ ID NO. 316,and 0.135 g NaNADP were added. With stirring at 22° C. and the automatictitrator prepared to provide 4N NaOH on demand, 270 g t-butyl6-cyano-(5R)-hydroxy-3-oxohexanoate was added from the addition funnel.The automatic titrator maintained the pH at 6.9 by the addition of 4NNaOH, which was continuously recorded. After 22 hours, 196.7 mL 4 N NaOHhad been consumed, and an extract of a sample analyzed by the method ofExample 4 showed >99% conversion. 450 mL ethyl acetate was added and themixture was filtered through diatomaceous earth. The aqueous phase wasseparated and extracted with 450 mL ethyl acetate. The solvent wasremoved from the combined organic extracts by rotary evaporation undervacuum to obtain 232 g crude t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoateas an oil.

This Example illustrates a larger scale preparation and recovery of asyn 3,5-dihydroxyhexanoate ester using an engineered ketoreductase bythe method of the invention.

8.20 Example 20 Reduction of t-butyl 6-cyano-(5S)-hydroxy-3-oxohexanoate

The S-enantiomer of t-butyl 6-cyano-5-hydroxy-3-oxohexanoate wasprepared from ethyl 4-cyano-(3S)-hydroxybutyrate (98% e.e.) following aprocedure analogous to the preparation of the R-enantiomer from ethyl4-cyano-(3S)-hydroxybutyrate described in Example 3.

To a 25 mL three-neck vessel equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into thevessel, was charged 20 mL 100 mM triethanolamine (chloride) buffer, pH7, 1.80 g glucose, 9 mg glucose dehydrogenase powder, 30 mgketoreductase powder comprising the engineered ketoreductase having theamino acid sequence SEQ ID NO. 316, 2.5 mg NaNADP, and lastly 3 gt-butyl 6-cyano-(5S)-hydroxy-3-oxohexanoate. The reaction mixture wasstirred at 22° C. The automatic titrator maintained the pH at 7 by theaddition of 4N NaOH, which was continuously recorded. Reaction progresswas monitored by the rate and cumulative addition of the base andperiodic sampling of the reaction mixture for extraction and analysis bythe method of Example 4. After 38 hours, the conversion was 47%. Thereaction was extracted with twice with 10 mL ethyl acetate. The combinedorganic extract was filtered through diatomaceous earth and the solventwas removed by rotary evaporation under vacuum to leave an oil.

Crude t-butyl 6-cyanomethyl-2,2 dimethyl-1,3-dioxane-4-acetate wasobtained from the oil (Example 7) and analyzed by the method of Example6, showing 95.3% d.e. (4R,6S) diastereomer, corresponding to the((3R,5S) diastereomer of the diol product of the reduction reaction.(Note: The (4R,6S) diastereomer has the same retention time as its(4S,6R) enantiomer, and the (4S,6S) diastereomer has the same retentiontime as its (4R,6R) enantiomer.)

When the engineered ketoreductase of this example is used to reduce theR-enantiomer of t-butyl 6-cyano-5-hydroxy-3-oxohexanoate, it providesthe syn (3R,5R) diol in essentially perfect diastereoselectivity (seeExamples 13, 14, and FIG. 6 and FIG. 7) This example illustrates that itis also 3R-selective when used to reduce the 5S-enantiomer of t-butyl6-cyano-5-hydroxy-3-oxohexanoate.

8.21 Example 21 Reduction of a 97% 5R, 3% 5S mixture of enantiomers oft-butyl 6-cyano-5-hydroxy-3-oxohexanoate (94% e.e.)

The procedure was identical to the preceding Example with the exceptionthat 2.91 g t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate and 0.09 gt-butyl 6-cyano-(5S)-hydroxy-3-oxohexanoate were added to the reaction.The reaction reached 97% conversion in 20 hours, after which point theproduct oil was recovered as in the preceding example.

Crude t-butyl (4,6)-6-cyanomethyl-2,2 dimethyl-1,3-dioxane-4-acetate wasobtained from the oil (Example 7) and analyzed by GC/MS by the method ofExample 6, showing a mixture of 99.73% (4R,6R) diastereomer,corresponding to the syn-(3R,5R) diastereomer of the diol, and 0.27% ofthe (4R,6S) diastereomer, corresponding to the (3R,5S) diastereomer ofthe diol (i.e., 99.46% d.e.).

In combination with the preceding Example, this Example illustrates anengineered ketoreductase having stereospecificity for reduction of theR-enantiomer of t-butyl 6-cyano-5-hydroxy-3-oxohexanoate over theS-enantiomer. The stereopurity at the 5-position of the (3R,5)-diol (andthe 6-position of the (4R,6)-dioxane derived therefrom) is substantiallyupgraded compared to that of the 5-hydroxy-3-oxohexanoate substrate,99.46% d.e. vs. 94% e.e. The engineered ketoreductase reacted less thanone tenth of the 3% S-substrate added to the reaction, while reactingessentially all of the 97% R-substrate. Thus, engineered ketoreductasesof the present disclosure can provide a higher degree of stereopurity inthe syn-3,5-dihydroxyhexanoate product when the 5-hydroxy-3-oxohexanoatesubstrate is of lower stereopurity. Further, when the5-hydroxy-3-oxohexanoate substrate is prepared from a 3-hydroxybutyrateester, as exemplified in Example 3, a higher degree of stereopurity inthe syn-3,5-dihydroxyhexanoate product can be obtained, in net, from3-hydroxybutyrate ester of lower stereopurity.

8.22 Example 22 Diastereopurity of t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate Prepared by the Method of theInvention

Crude t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate was prepared as an oilby the method of Example 17. A sample of the oil was dissolved at 1.0mg/mL in 9% acetonitrile in water and analyzed by the LC/MS/MS method ofExample 5. The chromatogram is shown, blown up in scale in FIG. 6, upperpanel. No diastereomer of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate wasdetected above 0.005%.

For comparison, a solution of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoatewas prepared from crystalline t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate obtained froma commercial source (Aldrich Cat#53, 901-5), containing 0.1% of the(4S,6R) diastereomer (analyzed by the method of Example 6; see Example23). 250 mg of the commercial t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate was suspendedin 2 mL 1:1 methanol:water containing 200 μL 1N HCl. The mixture wasstirred for 16 hours at room temperature. 500 μL saturated aqueoussodium bicarbonate and 5 mL of ethyl acetate were added. The organicphase was separated and solvents were removed by rotary evaporationunder vacuum to yield 187 mg t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoateas a slightly yellow oil.

A sample of this oil was dissolved at 1.0 mg/mL in 9% acetonitrile inwater and also analyzed by the LC/MS/MS method of Example 5. Thechromatogram is shown, blown up in scale, in FIG. 6, lower panel. Thechromatogram shows the peak for the 0.1% (3S,5R) diastereomer of t-butyl6-cyano-3,5-dihydroxyhexanoate resulting from the 0.1% of the (4S,6R)diastereomer in the commercial t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate.

This example, by comparison of the upper and lower chromatograms in FIG.6, illustrates the process of the invention providing a substantiallydiastereomerically pure syn 3,5-dihydroxyhexanoate compound.

8.23 Example 23 Diastereopurity of Uncrystallized t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate Prepared bythe Method of the Invention

Crude t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate prepared from ethyl4-cyano-(3R)-hydroxybutyrate of >99.9% e.e. in Example 3 was convertedto t-butyl (6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate,which was analyzed for diastereopurity by GC/MS prior tocrystallization.

To a 100 mL three-neck flask equipped with a PTFE-coated magneticstirring bar and a pH electrode connected to an automatic titrator forpH-controlled addition of base on-demand via a feeding tube into theflask, was charged 18 mL 100 mM triethanolamine (chloride) buffer, pH7.0, 12 g crude t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate, and 7.99 gglucose. The pH of the solution was readjusted to 7.0 with 0.26 mL 4NNaOH. 1 mL of the same buffer containing 200 mg ketoreductase powdercomprising the engineered ketoreductase having the amino acid sequenceSEQ ID NO. 316, 15 mg glucose dehydrogenase powder, and 5 mg NaNADP wasadded and the reaction mixture was stirred at 22° C. The automatictitrator maintained the pH at 6.9 by the addition of 4N NaOH, which wascontinuously recorded. An additional 15 mg glucose dehydrogenase powderand 1 g glucose were added after 4.6 hours and an additional 15 mgglucose dehydrogenase powder and 1.5 mg NaNADP were added after 7 hours.After a total of 12 hours 9.04 ml 4 N NaOH had been consumed, and anextract of a sample analyzed by the method of Example 4 showed 99.3%conversion. 2.5 g diatomaceous earth was added to the reaction mixture,which was then extracted three times with 50 mL ethyl acetate. Thesolvent was removed from the combined organic extracts by rotaryevaporation under vacuum to obtain 10.40 g crude t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate as an oil.

To the crude t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate was added 22 mL2,2-dimethoxypropane and 100 μL methanesulfonic acid. The mixture wasstirred at for 2 hours at room temperature. After addition of 5 mLsaturated aqueous sodium bicarbonate, the pH of the aqueous phase was8.0. The mixture was extracted with 60 mL hexane, and the solvent wasremoved from the extract by rotary evaporation under vacuum to leave agolden oil comprising t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate.

A sample of the oil was dissolved in ethyl acetate to 5 mg/mL, which wasanalyzed by the following GC/MS method:

Column: HP5MS (0.25 mm i.d., 25 μm df, 30 m)

Carrier gas: Helium, 9.44 psi, 0.7 mL/minute.

Oven: 50° C. for 1 min, then 30° C./min to 220° C., then hold at 220° C.for 3 min.

Retention Times: (4S,6R) 7.9 min; (4R,6R) 8.0 min.

Detection: single ion monitoring, m/z 198.

The GC/MS analysis showed the t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate oil to besubstantially diastereomerically pure. For comparison a sample ofcommercially obtained (Aldrich, catalog no. 53, 901-5), crystallinet-butyl (6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate wasdissolved in ethyl acetate to 5 mg/mL, and likewise analyzed, showing0.10% of the (4S,6R) diastereomer. FIG. 7 shows the overlapped GC/MSchromatograms of the oil and the crystalline t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate.

The oil was then dissolved in 17 mL hexane and 50° C., and the solutionwas cooled to 0° C. for crystallization. Filtration and drying in airyielded 8.1 g crystalline t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate of 98.7 area %chemical purity.

This example illustrates the process of the invention providing asubstantially diastereomerically pure protected syn3,5-dihydroxyhexanoate compound without any diastereomeric purification,for example, by crystallization.

8.24 Example 24 Preparation of t-butyl(6R)-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate (Formula V)

t-butyl (6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate, suchas prepared in Examples 18 and 23, is converted to the title compoundusing the methodology disclosed in U.S. Pat. No. 5,003,080 at column 49,lines 16-43, which is incorporated herein by reference. In particular, asolution of t-butyl(6R)-(2-cyanoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate, 5.63 g (0.048mol), in 100 mL of methanol saturated with gaseous ammonia is treatedwith 0.5 g of Raney nickel #30 and hydrogen gas in a shaker at 50 psiand 40° C. After 16 hours, thin layer chromatography indicates that nostarting nitrile present. The suspension is cooled, filtered through afilter aid, and concentrated to an oil. This crude oil is purified byflash chromatography on silica gel with 30:20:1 (ethylacetate:methanol:ammonium hydroxide) as eluent to give 4.93 g of thetitled compound: t-butyl(6R)-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate of Formula V(98.2 area %), as a clear oil with acceptable IR, NMR, C-NMR and MSspectra.

8.25 Example 25 Preparation of t-butyl(6R)-[2[2-(fluorophenyl)-5-(-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrol-1-yl]ethyl]-2,2-dimethyl-1,3-dioxane-(4R)-acetate(Formula VI)

t-butyl (6R)-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate, suchas prepared in Example 24, is converted to the title compound using themethodology disclosed in U.S. Pat. No. 5,003,080 at column 49, lines43-60, which is incorporated herein by reference. In particular, asolution of t-butyl(6R)-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-(4R)-acetate (formula IV),1.36% (4.97 mol), and(±)-4-fluoro-α-[2-methyl-1-oxopropyl]-γ-oxo-N,β-diphenylbenzenebutaneamide(formula V), which is a mixture of [R—(R′,R′)], [R—(R′,S′)], [S—(R′,R′)]and [S—R′,S′)]isomers, 1.60 g (3.83 mol), in 50 mL of heptane:toluene(9:1) is heated at reflux for 24 hours. The solution is cooled slightlyand 15 mL of 2-propanol added. The mixture is allowed to cool to 25° C.and filtered to give 1.86 g of the titled compound: t-butyl(6R)-[2[2-(fluorophenyl)-5-(-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrol-1-yl]ethyl]-2,2-dimethyl-1,3-dioxane-(4R)-acetateof formula VI, as a yellow solid with acceptable NMR spectra.

8.26 Example 26 Preparation of(2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-3-carboxamide(a.k.a. Atorvastatin Lactone) (Formula VII)

t-butyl(6R)-[2[2-(fluorophenyl)-5-(4-(1-methylethyl)-3-phenyl-4-[(phenylamino)-carbonyl]-1H-pyrrol-1-yl]ethyl]-2,2-dimethyl-1,3-dioxane-(4R)-acetate,such as prepared in Example 25, is converted to the title compound usingthe methodology disclosed in U.S. Pat. No. 5,003,080 at col. 50, lines4-30, which is incorporated herein by reference. In particular, 4.37 g(6.68 mol) of t-butyl(6R)-[2[2-(fluorophenyl)-5-(-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrol-1-yl]ethyl]-2,2-dimethyl-1,3-dioxane-(4R)-acetateis dissolved in 200 mL of tetrahydrofuran and 15 mL of 10% hydrochloricacid solution is added, and the solution is stirred for 15 hours. Tothis solution is added sodium hydroxide (3.6 g) and the mixture isstirred for 30 hours. The reaction is stopped by adding 150 mL of water,90 mL of hexane, and separating the layers. The aqueous layer isacidified with dilute hydrochloric acid solution, stirred for threehours and extracted with 150 mL of ethyl acetate. A drop of concentratedhydrochloric acid is added to the ethyl acetate solution and thesolution is allowed to stand 18 hours. The solution is concentrated invacuo and the concentrate is redissolved in 50 mL of ethyl acetate andtreated with one drop of concentrated hydrochloric acid. The solution isstirred two hours, concentrated in vacuo, and dissolved in 3.0 mL oftoluene. The titled compound,(2R-trans-5-(4-fluorophenyl)-2-(1-methyl-ethyl)-N,4-diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-3-carboxamide(3.01 g) of Formula VII, which is also known as atorvastatin lactone, isisolated in two crops.

8.27 Example 27 Preparation of[R—(R*,R*)]-2-(4-fluorophenyl)-β-δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoicacid, Calcium Salt (2:1) of Formula VIII

(2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-3-carboxamide,such as prepared in Example 26, is converted to the title compound ofFormula VIII using the methodology disclosed in Examples 1A or 1B ofU.S. Pat. No. 5,969,156, entitled “Crystalline[R—(R*,R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoicacid hemi calcium salt (atorvastatin),” which issued on Oct. 19, 1999and which examples are incorporated herein by reference. The titlecompound is well known under the generic name atorvastatin, hemi calciumsalt and the brand name LIPITOR®. Preparation of the calcium salt fromthe lactone of Example 26 is disclosed in Example 10 of U.S. Pat. No.5,273,995 which is incorporated herein by reference.

i. Sodium Salt

In particular, dissolve one mole of the lactone (540.6 g) from Example26 in 5 L of methanol. After dissolution, add 1 L of H₂O. Whilestirring, add 0.98 equivalents NaOH and follow the reaction by HPLCuntil 2% or less lactone and methyl ester of the diol acid remains. Anexcess of NaOH is not used because Ca(OH)₂ can form upon the addition ofCaCl₂ in a subsequent step. Upon hydrolysis of the lactone,[R—(R*,R*)]-2-(4-fluorophenyl)-β-δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoicacid, sodium salt (atorvastatin, sodium salt) is formed.

ii. Calcium Salt

Upon completion of hydrolysis, add 10 L H₂O, then wash at least twotimes with a 1:1 mixture of EtOAc/hexane. Each wash should contain 10 Leach of EtOAc/hexane. If sodium salt is pure, add 15 L of methanol. Ifit is impure and/or contains color, add 100 g of G-60 charcoal, stir fortwo hours, filter over supercel and then wash with 15 L MeOH. Perform ananalysis on the reaction mixture by HPLC to determine the exact amountof the sodium salt of atorvastatin that is in the aqueous methanolsolution.

Dissolve 1 equivalent or slight excess of CaCl₂.2H₂O (73.5 g) in 20 LH₂O. Heat both the CaCl₂ solution and the aqueous methanol solution to60° C. Add the CaCl₂ solution slowly, with high agitation to the 60° C.aqueous methanol solution of atorvastatin, sodium salt. After completeaddition, cool the mixture slowly to 15° C. and filter the precipitatingcalcium salt of formula VIII. Wash the filter cake with 5 L H₂O. Dry at50° C. in a vacuum oven.

The calcium salt from the dried filter cake can be recrystallized bydissolving in 4 L of EtOAc (50° C.), filtering over Supercel®, washingthe filtrate with 1 L EtOAc (50° C.), then adding 3 L of a nonpolarsolvent such as hexane to the 50° C. EtOAc solution. The resultingcrystals of formula VIII are then dried by air or with heating.

8.28 Example 28 Production of Ketoreductases; Fermentation Procedure

In an aerated agitated 15 L fermenter, 6.0 L of growth medium containing0.88 g/L ammonium sulfate, 0.98 g/L of sodium citrate; 12.5 g/L ofdipotassium hydrogen phosphate trihydrate, 6.25 g/L of potassiumdihydrogen phosphate, 6.2 g/L of Tastone-154 yeast extract, 0.083 g/Lferric ammonium citrate, and 8.3 ml/L of a trace element solutioncontaining 2 g/L of calcium chloride dihydrate, 2.2 g/L of zinc sulfateseptahydrate, 0.5 g/L manganese sulfate monohydrate, 1 g/L cuproussulfate heptahydrate, 0.1 g/L ammonium molybdate tetrahydrate and 0.02g/L sodium tetraborate decahydrate was brought to a temperature of 30.The fermenter was inoculated with a late exponential culture of E. coliW3110, containing a plasmid with the ketoreductase gene of interest,grown in a shake flask as described in Example 3 to a starting OD₆₀₀ of0.5 to 2.0. The fermenter was agitated at 500-1500 rpm and air wassupplied to the fermentation vessel at 1.0-15.0 L/min to maintain adissolved oxygen level of 30% saturation or greater. The pH of theculture was controlled at 7.0 by addition of 20% v/v ammonium hydroxide.Growth of the culture was maintained by the addition of a feed solutioncontaining 500 g/L cerelose, 12 g/L ammonium chloride and 10.4 g/Lmagnesium sulfate heptahydrate. After the culture reached an OD₆₀₀ of50, the expression of ketoreductase was induced by the addition ofisopropyl-b-D-thiogalactoside (IPTG) to a final concentration of 1 mM.The culture was grown for another 14 hours. The culture was then chilledto 4° C. and maintained at 4° C. until harvested. Cells were harvestedby centrifugation at 5000 G for 40 minutes in a Sorval RC12BP centrifugeat 4° C. Harvested cells were used directly in the following downstreamrecovery process or were stored at 4° C. until such use.

The cell pellet was resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 6.8, at 4° C. to each volume of wet cell paste.The intracellular ketoreductase was released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate was cooled to 4° C. immediately after disruption. A solutionof 10% w/v polyethyleneimine, pH 7.2, was added to the lysate to a finalconcentration of 0.5% w/v and stirred for 30 minutes. The resultingsuspension was clarified by centrifugation at 5000 G in a standardlaboratory centrifuge for 30 minutes. The clear supernatant was decantedand concentrated ten fold using a cellulose ultrafiltration membranewith a molecular weight cut off of 30 Kd. The final concentrate wasdispensed into shallow containers, frozen at −20° C. and lyophilized topowder. The ketoreductase powder was stored at −80° C.

This Example illustrates the production of ketoreductase powders by apractical scaleable procedure.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. An engineered or isolated ketoreductase enzyme capable ofstereoselectively reducing the 3-oxo group of a 5-hydroxy-3-oxohexanoateto yield the corresponding syn 3,5-dihydroxyhexanoate ester and havingincreased specific activity as compared to the wild-type ketoreductaseenzyme of SEQ ID NO:2, wherein the ketoreductase enzyme comprises anamino acid sequence that is at least 90% identical to SEQ ID NO: 2 andcomprises at least one mutation relative to SEQ ID NO: 2 that is X¹⁶⁰ isT.
 2. The ketoreductase of claim 1, wherein the specific activity of theketoreductase is at least about 1.5 or greater than the specificactivity of the wild-type enzyme of SEQ ID NO:
 2. 3. The ketoreductaseenzyme of claim 1, which comprises an amino acid sequence that has oneor more of the following characteristics: X⁶ is a polar or acidic aminoacid residue; X²⁷ is an aliphatic or basic amino acid residue; X³¹ is ahydroxyl containing or aliphatic amino acid residue; X³⁶ is a smallamino acid residue; X⁴⁸ is a basic amino acid residue; X⁶³ is ahydrophobic or aromatic amino acid residue; X⁶⁵ is a hydroxyl containingor small amino acid residue; X⁸⁶ is an aliphatic or hydrophobic aminoacid residue; X¹²⁵ is an aliphatic or aromatic amino acid residue; X¹⁵²is a basic amino acid residue; X¹⁶⁵ is a polar or acidic amino acidresidue; X¹⁹⁴ is an aromatic amino acid or polar hydrophilic amino acidresidue X²¹⁴ is a polar hydrophilic amino acid residue; X²¹⁸ is ahydroxyl containing amino acid residue; X²³⁴ is a polar hydrophilicamino acid residue; X²⁴⁸ is an aliphatic amino acid residue; X²⁵⁰ is abasic amino acid residue; X²⁶³ is a acidic or polar amino acid residue;X²⁹⁰ is an acidic amino acid residue; X²⁹⁶ is an aliphatic amino acidresidue X²⁹⁷ is an aromatic amino acid residue; X³⁰¹ is an aliphatic orhydroxyl containing or acidic or hydrophilic or basic amino acidresidue; and X³⁰⁷ is an aromatic, a polar or a hydroxyl containing aminoacid residue.
 4. The ketoreductase enzyme of claim 3 which includes oneor more mutations selected from: X⁶ is E; X²⁷ is A or K; X³¹ is S or P;X³⁶ is V; X⁴⁸ is H; X⁶³ is W; X⁶⁵ is G or S; X⁸⁶ is I; X¹²⁵ is L; X¹⁵²is R; X¹⁶⁵ is E X¹⁹⁴ is C; X²¹⁴ is R or H; X²¹⁸ is T; X²³⁴ is Q; X²⁴⁸ isI; X²⁵⁰ is R; X²⁶³ is E; X²⁹⁰ is V; X²⁹⁶ is I; X²⁹⁷ is F or W; X³⁰¹ isR, K, A, V, S, T, Y, D, E, P, or Q; and X³⁰⁷ is H, N or S.
 5. Theketoreductase enzyme of claim 1 which comprises an amino acid sequenceselected from SEQ ID NOS: 18, 54, 56, 66, 72, 82, 84, 92, 94, 96, 98,100, 104, 106, 112, 114, 116, 118, 120, 122, 124, 126, 128, 136, 138,140, 142, 144, 146, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, and366.
 6. A process for preparing a syn 3,5-dihydroxyhexanoate compoundaccording to formula (IIa):

wherein X is selected from halo, cyano and —OR⁶, where R⁶ is hydrogen ora protecting group, and R¹ is selected from (C1-C12) alkyl, (C6-C10)aryl and (C7-C12) arylalkyl, comprising stereoselectively reducing anenantiomer of a 5-hydroxy-3-oxohexanoate compound according tostructural formula (Ia):

wherein X and R¹ are as defined for structural formula (IIa); using aketoreductase according to claim 1 under conditions suitable forreducing the 3-oxo group to a 3-hydroxy group.
 7. The process of claim 6which is carried out with whole cells that express the ketoreductaseenzyme, or an extract or lysate of such cells.
 8. The process of claim 6in which the ketoreductase is isolated and/or purified and theenantioselective reduction reaction is carried out in the presence of acofactor for the ketoreductase and optionally a regeneration system forthe cofactor.
 9. The process of claim 6 in which X is selected fromchloro, cyano and hydroxyl, and in which R¹ is a lower alkanyl ort-butyl.
 10. The process of claim 8 in which the cofactor is NADH and/orNADPH, and in which the weight ratio of the cofactor to theketoreducatase enzyme is in the range of about 10:1 to 100:1.
 11. Theprocess of claim 8 in which the cofactor regenerating system comprisesglucose dehydrogenase and glucose; formate dehydrogenase and formate; orisopropanol and a secondary alcohol dehydrogenase.
 12. The process ofclaim 11 wherein the secondary alcohol dehydrogenase is theketoreductase.
 13. In a method of synthesizing a statin compound thatincludes as one step the enantioselective reduction of a compoundaccording to structural formula (Ia) to yield a compound according tostructural formula (IIa), the improvement comprising carrying out theenantioselective reduction of compound (Ia) to compound (IIa) with aketoredutase enzyme according to claim 1; wherein structural formula(Ia) is:

wherein X is selected from halo, cyano and —OR⁶, where R⁶ is hydrogen ora protecting group, and R¹ is selected from (C1-C12) alkyl, (C6-C10)aryl and (C7-C12) arylalkyl; and wherein structural formula (IIa) is:

wherein X and R¹ are defined as for structural formula (Ia).
 14. Themethod of claim 13 in which the statin is selected from atorvastatin,rosuvastatin, and pitavastatin.